TR201807202T4 - CD70 antibodies. - Google Patents

Abstract

The present invention relates to antibodies and antigen binding fragments thereof that bind to human CD70 protein with high affinity and strongly inhibit tumor cell growth. The antibody or antigen binding fragment comprises at least one heavy chain variable domain (VH) comprising a variable heavy chain CDR3, a variable heavy chain CDR2 and a variable heavy chain CDR1, and a variable light chain CDR3, a variable light chain CDR2, and a variable. at least one light chain variable domain (VL) comprising the light chain CDR1.

Description

DESCRIPTION CD70 ANTIBODIES Technical Area The present invention binds to human CD70 protein with high affinity and is a tumor cell strongly inhibiting the growth of antibodies and their antigen-binding related to fragments.

Known Matters of the Invention Cytokine receptor CD27 tumor necrosis factor receptor (TFNR) superfamily, cell It is a member that plays a role in apoptosis along with its growth and differentiation. CD27 ligand, CD707, which belongs to the tumor necrosis factor family of ligands. CD70 is a 20 amino acid hydrophilic N-terminal domain (antibody domain with special function) and 2 potential N-linked It is a 193 amino acid polypeptide with a C-terminal domain containing a glycosylation domain. It has been identified as a transmembrane protein.

CD70 is briefly present in activated T and B lymphocytes and dendritic cells in addition to expression in cells, renal cell carcinomas, metastatic breast cancers, brain different cancers, including tumors, leukemias, lymphomas and nasopharyngeal carcinomas It has been reported that CD70 expression is also present in species (Junker et al., (2005) J Urol. It has also been suggested to play a role in mediated autoimmune disease and inhibition of TNF-alpha production. Thus, CD70 is associated with cancer, autoimmune disorders and CD70 expression. It constitutes a target for the treatment of a number of other diseases. are described, said antibodies to either ADCC, ADCP, CDC, or ADC9, or may mediate more and in a CD70-expressing cancer cytostatic or may exert a cytotoxic effect or be conjugated to a cytostatic or cytotoxic agent. An immunosuppressive effect in an immunological disorder expressing CD70 without they can display. The antibodies exemplified herein are two monoclonal antibodies named 1F6 and 2F2. humanized anti-CD70 antibodies, specifically humanized 1F6 and The humanized 2F2 is described. is explained. These antibodies are characterized by human CD703e with a KD of 1x10'7 M or less. are binding. Renal cell carcinoma, such as 786-0, expressing antibodies CD70 They also bind to and internalize tumor cell lines.

Summary of the Invention An antibody that binds to human CD70, or an antigen-binding component thereof, in accordance with the invention. fragment is provided and said antibody or antigen-binding fragment is a variable. most containing heavy chain CDR3, a variable heavy chain CDR2 and a variable heavy chain CDR1 a small heavy chain variable domain (VH) and a variable light chain CDR3, a variable light at least one light chain variable domain containing chain CDR2 and one variable light chain CDR1 (VL); where variable heavy chain CDR3 SEQ ID. NO:50 (DAGYSNHVPIFDS) or it consists of; variable heavy chain CDR2 SEQ ID. NO: contains or it consists of; variable heavy chain CDRl SEQ ID. NO2]1 (VYYMN); variable light chain CDR3 SEQ ID. NO: includes or consists of; variable light chain CDR2 SEQ ID 1D. NO: includes or consists of; and variable light chain CDR1 SEQ ID. NO: includes or Here, CD70 antibodies that bind to human CD70 protein and are described in the prior art antibodies or their antigens exhibiting different and generally superior properties Binder fragments (herein referred to as CD70 antibodies) are provided. These antibodies its superior properties in human treatment; cancers, particularly those expressing CD70, and also It provides advantages in terms of use in the treatment of immunological disorders.

The CD70 antibodies described herein feature exceptionally high binding for human CD70. show affinity. All preferred embodiments described herein are recoinbinant human Of the most potent CD70 antibodies of the prior art recommended in human therapy for CD70, for example, significantly greater than prior art "fully human" CD70 antibodies a higher binding affinity (BiacoreTM surface plasmon as described here measured by resonance). In addition, preferred CD70 antibodies disclosed herein all embodiments with prior art CD70 antibodies recommended for human therapy. Compared to human cell lines, specifically human cancer cell lines It exhibits superior (ie, high affinity) binding to CD70 expressed on the surface of the lines.

This superior binding to CD70 on the cell surface is particularly important for CD70°i "low copy". prominent in human cancer cell lines expressing "number of directly related to its use in human therapy. Also, the CD70 described here Preferred embodiments of antibodies have been previously proposed for therapeutic use in humans. cancer isolated from human patients as compared with prior art CD70 antibodies cancer cells isolated from patients with chronic lymphocytic leukemia (CLL) They exhibit significantly enhanced binding to their cells.

An antibody or an antigen-binding fragment thereof that binds to human CD70. disclosed, wherein said antibody or antigen-binding fragment has at least one heavy chain variable domain (VH) and at least one light chain variable domain (VL), mentioned here The subject VH and VL domains also use the standard BiacoreTM protocol described here. less than 7 x 10'4 s for human CD70 when tested as a Fab fragment speed (cone measured by BiacoreTM).

The antibody or antigen-binding fragment has at least one heavy chain variable domain (VH) and at least may comprise a light chain variable domain (VL), wherein said VH and VL It exhibits a separation rate of s or less. Most preferably the CD70 antibody is a Fab fragment will display.

An antibody that binds to human CD70, in which the antibody in question contains two Fab regions, is also is also described, wherein each of the Fab regions binds to human CD70 and is a Fab exhibits a rate of separation. Two Fab regions may be identical or have binding properties, eg. may differ for human CD70 in terms of atinitiation. The two Fab regions are identical in human CD70. may bind to the epitope or overlapping epitopes, or to separate, non-overlapping epitopes in human CD70. can be attached. Amino acid in one or both of the two Fab regions VH and VL domains may differ from each other in terms of sequence.

Preferred embodiments of CDO antibodies presented here are exceptionally high for CD70. In addition to binding affinity, strong blockade of the interaction between CD70 and its ligand CD27 or inhibition. Both CD70 bind with high affinity, and CD70/CD27 Preferred CD70 antibodies exhibit strong blockade of interaction, CD70/CD27 signaling potentiates the therapeutic efficacy (for example, CD70 antibody in addition to cell death mediated by effector functions) eg CD70 and CD27° disease in diseases such as autoimmune diseases and cancers in which it is co-expressed They are particularly advantageous as therapeutic agents for the treatment of

All of the CD70 antibodies described herein, in addition to binding to CD70 with high affinity, It does not exhibit strong blockade in the CD70/CD27 interaction. Here CD709e has very high affinity. binding but not significantly blocking the CD70/CD27 interaction some CD70 antibodies are also described. The characteristics of these antibodies are elsewhere here. is explained. The presence of high-affinity non-blocking CD70 antibodies can increase/expand the range of possibilities.

CD70 described here exhibiting very high binding affinity for human CD70 Preferred embodiments of antibodies have been proposed for therapeutic use in humans. have a combination of binding properties not exhibited by prior art CD70 antibodies They also show the feature of being. Thus, the preferred CD70 antibodies disclosed herein It has the following features: (a) binding with the human CD70ite amino acid sequence HlQVTLA1CSS (SEQ ID NO:342); (b) rhesus macaque (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus) cross-reactivity with CD70 homologues; (c) both natural human CD70e and heat-denatured recombinant human CD70e don't bind.

This is not exhibited by prior art antibodies recommended for therapeutic use in humans. combination of binding properties in CD70 than prior art CD70 antibodies It indicates binding to a new epitope different from the epitopes to which it binds.

The combination of binding properties exhibited by preferred CD70 antibodies to human advantageous for drug development. Specifically, with monkey CD70 homologues cross-reactivity related to CD70 antibodies proposed for therapeutic use in humans makes it possible to carry out toxicology studies in primate models.

The preferred CD70 antibodies disclosed herein are also used as a therapeutic antibody product. exhibit positive Properties associated with commercial manufacturing. Explained elsewhere here The preferred CD70 antibodies presented herein are therapeutic antibodies suitable for clinical use. outstanding in recombinant expression systems used in the commercial manufacture of products exhibits high levels of expression. achievable with preferred CD70 antibodies expression levels typically obtained with "fully human" therapeutic antibody products. even higher than the levels. In addition, preferred CD70 antibody products (human produced by recombinant expression in a format suitable for therapeutic use) They exhibit an outstanding thermal stability superior to antibody products.

It has superior binding affinity for human CD70 and other advantageous properties listed above. CD70 antibodies presented herein are from camelids (eg, from llamas).

CD70 antibodies from camelids were isolated or recombinantly expressed. may be monoclonal antibodies. Preferred embodiments are humanized (or germline a processed ("germlined") monoclonal antibody (for example, a camelid a humanized variant of the antibody), a chimeric antibody (for example, a camelid-human chimeric antibody) or a humanized chimeric antibody (e.g. camelid VH and VL domains) a chimeric containing humanized variants and constant domains of a human antibody. antibodies).

Camellia-derived CD70 antibodies have a heavy chain variable domain (VH) and light chain variable domain (VL), where the VH and VL domains are derived from camelids. In particular embodiments, the antibody or antigen-binding fragment does not represent the VH and VL domains. or human germline processed variants of the llama VH and VL domains.

This antibody or antigen-binding fragment is "high human homology" as described herein. can display.

The camelid-derived CD70 antibodies described here are typically closest match. exhibits.

Other preferred embodiments of the invention are camelid-derived CD70 antibodies. includes humanized (or human germline-processed) variants. In particular, the invention Humanized or human germ cells of CD70 antibodies from the llama described herein Includes line-processed variants.

In another aspect of the invention, a chimeric camelid-human antibody that binds to human CD70 provided, wherein the antigen-binding portions of the antibody (for example, VH and/or VL) domains or their CDRs) are derived from camelids and the constant regions of the antibody are derived from human antibody. In particular, a chimeric lama that binds to human CD70 in the invention In another aspect of the invention, a chimeric camel-human binding to human CD707 A humanized variant of the antibody is provided, wherein the antibody is portions (for example, the VH and/or VL domains or their CDRs) are obtained from camelids. are humanized variants of the extracted sequences and the constant regions of the antibody are obtained from the antibody. Specifically, a chimeric lama-human binding to human CD705 in the invention A humanized variant of the antibody is provided.

Here below specific structural features, namely CDRs (SEQ ID: NO: 49-59, 262 or SEQ ID: NO: one or more or one of the CDR sequences shown as CDR1) or all of the variable domains (SEQUENTIAL) or one or more of 277 (VL) with reference to the specified amino acid sequences CD70 antibodies or antigen-binding fragments thereof that have been identified are described. This all antibodies bind with high affinity to human CD70, tested as an F ab fragment They exhibit a separation rate of '4 s`l.

Here, specifically, VH and VL domains obtained from camelids or their human antibodies to the constant domains of human IgG1, IgG2, IgG3 or IgG47 chimeric antibodies containing fused human germline-treated variants is provided. Heavy chain variable domains single-domain antibodies described above They can be used as a conventional four-chain antibody or other antigen. binding proteins, eg Fab, Fab', F(ab')2, bi-specific Fabs and FV fragments, di- antibodies, linear antibodies, single-chain antibody molecules, single-chain variant fragment (scFv) and multispecific antibodies.

A binding site for any given CD70 antibody or its CD70 antigen antigen binding that contains a VH domain paired with a VL domain to form fragment contains a combination of 6 CDRs: variable heavy chain CDR3 (HCDR3), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDRS), variable light chain CDR2 (LCDR2) and variable light chain CDR1 Preferred combinations of 6 CDRs include: (i) SEQ ID. HCDR3 containing NO:50, SEQ ID. HCDRZ containing NO:27, SEQ ID. containing LCDR2 and SEQ ID. LCDR1 containing NO:250; Here, variable heavy chain CDR3 (HCDR3), variable heavy chain CDR2 (HCDRZ), variable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3), variable light chain Combinations of CDR2 (LCDR2) and variable light chain CDR] (LCDR1) are also disclosed. are selected from the group consisting of: (ii) SEQ ID. HCDR3, including NO:49, SEQ ID. HCDRZ containing NO:265, SEQ ID. containing LCDR2 and SEQ ID. LCDR1 containing NO:77°; (iii) SEQ ID. HCDR3, including NO:50, SEQ ID. HCDRZ containing No:27, SEQ ID.

HCDRI containing No:1 1, SEQ ID. LCDR3, including No:199, SEQ ID. NO:1 10 containing LCDR2 and SEQ ID. LCDRI containing NO:78; (iv) SEQ ID. HCDR3, including NO:50, SEQ ID. HCDRZ containing No:28&, SEQ ID.

HCDR1 containing NO: 11, SEQ ID. LCDR3, including NO:150°, SEQ ID. NO] 1 1”i containing LCDR2 and SEQ ID. LCDR1 containing NO:797u; (v) SEQ ID. HCDR3, including NO:50, SEQ ID. HCDR2 with NO:28, SEQ ID.

HCDR1 containing NO:11, SEQ ID. LCDR3, including NO: 15 1, SEQ ID. NO:110°u containing LCDR2 and SEQ ID. LCDR1 containing NO:80°i; (Vi) SEQ ID. HCDR35 SEQ ID containing NO:51. HCDRZ containing NO:29, SEQ ID.

HCDRI containing NO:12, SEQ ID. LCDR3, including NO: 152°, SEQ ID. NO:110 containing LCDR2 and SEQ ID. LCDRI containing NO:80; (vii) SEQ ID. HCDR3, including NO:52, SEQ ID. HCDR2 containing NO:30, SEQ ID.

HCDRI containing NO:13, SEQ ID. LCDR3, with NO:153, SEQ ID. NO:I 12,yi containing LCDR2 and SEQ ID. LCDRI containing NO:81; (viii) SEQ ID. HCDR3, including NO:53, SEQ ID. HCDR2 with NO:31°i, SEQ ID.

HCDR1 containing NO: 14, SEQ ID. LCDR3, including NO: 154, SEQ ID. NO:1 13°ü containing LCDR2 and SEQ ID. LCDR1 containing NO:82; (ix) SEQ ID. HCDR3, including NO:54, SEQ ID. HCDR2 with NO:32, SEQ ID. containing LCDR2 and SEQ ID. LCDRI containing NO:83°; (x) SEQ ID. HCDR3, including NO:55”, SEQ ID. HCDR2 with No:3311, SEQ ID. containing LCDR2 and SEQ ID. LCDRI containing NO:84; (xi) SEQ ID. HCDR3, SEQ ID containing N0:56*. HCDR2 with No:3411, SEQ ID.

HCDR1 containing NO:17, SEQ ID. LCDR3, including NO: 157*, SEQ ID. No:116°yi containing LCDR2 and SEQ ID. LCDR1 containing NO:85,i; (xii) SEQ ID. HCDR3, including NO:57, SEQ ID. HCDR2 with NO:35, SEQ ID.

HCDR1 containing NO: 18, SEQ ID. LCDR3, with NO: 158, SEQ ID. NO:117,yi containing LCDR2 and SEQ ID. LCDR1 containing NO:84; (xiii) SEQ ID. HCDR3, with NO:583, SEQ ID. No:36 well containing HCDR2, SEQ ID.

HCDRI containing NO:19, SEQ ID. LCDR3, including NO: 159, SEQ ID. NO:118”i containing LCDR2 and SEQ ID. LCDR1 containing NO:86; (xiV) SEQ ID. HCDR3, including NO: 50, SEQ ID. HCDR2 with NO: 27, SERIAL containing LCDR2 and SEQ ID. LCDR1 containing NO:88”; (xv) SEQ ID. HCDR3, including NO: 50, SEQ ID. HCDR2, including NO: 27, SEQ ID.

NO: 11, containing HCDRI, SEQ ID. LCDR3, including NO:162, SEQ ID. NO: 121,i containing LCDR2 and SEQ ID. LCDRI containing NO:89; (xvi) SEQ ID. HCDR3, including NO: 50, SEQ ID. HCDR2 with NO: 277, SEQUENCE ID. HCDRI containing NO: 11,I, SEQ ID. LCDR3, including NO: 163, SEQ ID. NO: 122°yi containing LCDR2 and SEQ ID. LCDRI with NO: 9071; (xvii) SEQ ID. HCDR3 with NO: 51>i SEQ ID. HCDR2 with NO: 297u, SEQ ID.

NO: 12 well containing HCDR1, SEQ ID. LCDR3, including NO: 164, SEQ ID. NO: 123” containing LCDR2 and SEQ ID. LCDR1 containing NO: 91”; (xviii) SERIAL 1D. HCDR3, with NO: 517i, SEQ ID. HCDR2 containing NO: 29, SEQ ID.

HCDR1 containing NO: 129, SEQ ID. LCDR3, including NO: 164, SEQ ID. NO: l24” containing LCDR2 and SEQ ID. LCDR1 containing NO: 91; (xix) SEQ ID. HCDR3, including NO: 59, SEQ ID. HCDR2 with NO: 373, SEQ ID.

HCDR1 containing well NO: 12, SEQ ID. LCDR3, with NO: 165, SEQ ID. NO: 125,i containing LCDR2 and SEQ ID. LCDR1 containing NO: 92*; (xx) SEQ ID. HCDR3, including NO: 59, SEQ ID. HCDR2 with NO: 377, SEQ ID.

HCDR1 containing NO: 20°, SEQ ID. NO: LCDR3, with 165°i SEQ ID. NO: 126” containing LCDR2 and SEQ ID. LCDRI containing NO: 93; (xxi) SEQ ID. HCDR3, including NO: 59, SEQ ID. HCDR2 containing NO: 37, SEQ ID. containing LCDR2 and SEQ ID. LCDR1 containing NO: 92”; (xxii) SEQ ID. HCDR3, including NO: 59, SEQ ID. NO: HCDR2 including 37°, SEQUENCE ID. HCDR1 containing NO: 207, SEQ ID. LCDR3, including NO: 167, SEQ ID. NO: LCDR2 and SEQ ID containing 128. LCDR1 containing NO: 94; (xxiii) SEQ ID. HCDR3, including NO: 59, SEQ ID. HCDR2 with NO: 377, SEQUENCE ID. HCDRI containing NO: 20, SEQ ID. LCDR3, including NO: 168, SEQ ID. NO: LCDR2 and SEQ ID containing 110”. LCDR1 containing NO: 951; (xxiV) SEQ ID. HCDRS containing NO: 262, SEQ ID. NO: HCDR2 containing 258%, SERIAL ID. HCDRI with NO: 2563y1, SEQ ID. NO: 271, LCDR3, SEQ ID. NO: (xxv) SEQ ID. HCDR3, including NO: 263°, SEQ ID. HCDR2, SEQ ID NO: 259 ID. HCDR1 containing NO: 257, SEQ ID. LCDR3, with NO: 2733, SEQ ID. NO: LCDR2 containing 270,i and SEQ ID. LCDR1 containing NO: 268.

Here we present isolated antibodies or antibodies that exhibit high affinity binding to human CD70. containing antigen-binding fragments thereof, and SEQ ID. NO: 177, SEQ ID. No:178, SERIAL at least 90%, 95%, 97%, 98% or , optionally SEQ ID, which contains a heavy chain variable domain with sequence.

NO:21 i, SEQ ID.

No:240, SERIAL lD NO:276 and SEQ ID. amino acid of NO:277° to an amino acid sequence selected from the group consisting of amino acid sequences exhibiting identity CD70 antibodies comprising a light chain variable domain with

The above listed VH and VL domain sequences of VH domains and VL domains Although all possible matches from selected groups are possible, some VH and VL combinations are particularly preferred; These are human CD70 with high affinity. are "natural" combinations within a single Fab that exhibit binding.

Thus, preferred CD70 antibodies exhibit high affinity CD70 binding. or their antigen-binding fragments have a heavy chain variable domain (VH) and a light chain variable domain (VL) combination, wherein the combination is is as specified: (i) SEQ ID. a heavy chain variable domain containing the amino acid sequence of NO:223 and SEQ ID. a light chain variable domain containing the amino acid sequence of NO:241; Here, a heavy chain variable domain (VH) and a light chain variable domain (VL) combination and the combination is selected from the group consisting of those listed below. CD70 antibodies or their antigen exhibiting high affinity CD70 binding connector fragments are also described: (ii) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:177 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO:189; (iii) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO: 178 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 19021; (iv) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO: 1793 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 191; (v) SEQ ID. a heavy chain variable domain containing the amino acid sequence of NO: 17921, and SEQ ID. a light chain variable domain containing the amino acid sequence of NO:192; (vi) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO: 180 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO:193; (vii) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:18] and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO:194; (viii) SEQUENCE 1D. A heavy chain variable containing the amino acid sequence of NO: 182 domain and SEQ ID. A light chain variable containing the amino acid sequence of NO: 195 domain; (ix) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:183 and SEQ ID. a light chain variable domain containing the amino acid sequence of NO: 1961; (x) SEQ ID. a heavy chain variable domain containing the amino acid sequence of NO:184, and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 197a; (xi) SEQ ID. A heavy chain variable domain containing the amino acid sequence of No:185 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO:198; (xii) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:186 and SEQUENCE 1D. a light chain variable domain containing the amino acid sequence of NO: 199; (xiii) SEQ ID. A heavy chain variable containing the amino acid sequence of NO:187 domain and SEQ ID. A light chain variable containing the amino acid sequence of No:200 domain; (xiv) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:178 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO:201; (XV) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:178 and SEQ ID. a light chain variable domain containing the amino acid sequence of NO:202; (xvi) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:178 and SEQ ID. No: 203? a light chain variable domain containing the amino acid sequence of (xvii) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:178 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO:204; (xviii) SEQ ID. A heavy chain variable containing the amino acid sequence of NO: 180 domain and SEQ ID. A light chain variable containing the amino acid sequence of NO:205° domain; (Xix) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO: 180 and SEQ ID. a light chain variable domain containing the amino acid sequence of No:206; (xx) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO: 188 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO:20T; (xxi) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:188 and SEQ ID. No: 208? a light chain variable domain containing the amino acid sequence of (xxii) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:188 and SEQ ID. a light chain variable domain containing the amino acid sequence of NO:209; (xxiii) SEQ ID. A heavy chain variable containing the amino acid sequence of NO:188 domain and SEQ ID. A light chain variable containing the amino acid sequence of NO:210 domain; (xxiv) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:18876 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO:211; (XXV) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:274 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO:276; (xxvi) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:275 and SEQ ID. A light chain variable domain containing the amino acid sequence of NO:277.

The VH domain mentioned in each of the specific VHN L combinations listed above It is also possible to combine it with a VL domain having the amino acid sequence. In the previous paragraph and elsewhere here, antibodies/antigen-binding fragments % of sequence identity, whose structure is with said reference sequence (with a given SEQ ID. NOisu) is defined. In this context, the % sequence identity between two amino acid sequences is optimal. can be calculated by comparing these two sequences aligned in some fashion, and where The amino acid sequence to be compared is to ensure an optimum alignment between these two sequences. may contain additions or deletions relative to the reference string. Identity percentage between two series determining the number of identical positions at which the amino acid residue is identical, dividing the number of locations by the total number of locations in the compare window, and Multiplying the result obtained by 100.1 and thus obtaining the percent identity between these two series. calculated by obtaining it. Typically, the comparison window identifies the sequence being compared. corresponds to the height. For example, on the website http://WWW.ncbi.nlm.nih.gov/gorf/b12.html of the current BLAST program, "BLAST 2 sequences" (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol are given as (especially "gap penalty": 5 and "space extension penalty": 2 for parameters; selected matrix is for example the "BLOSUM 62" matrix suggested by the program), this The percent identity between the two strings compared in the figure is calculated directly by the program.

It is a particularly advantageous one, including binding to human CD70 with exceptionally high affinity. The most preferred CD70 antibodies presented herein exhibiting a combination of properties, Antigen-binding Fab from the llama designated 27B3 in the accompanying examples plus human germline-treated variants of 27B3, for example in the accompanying are antibodies based on the germline-processed variants described in the examples. 27B3 and germline-operated variants thereof, particularly CDRs of variant 4ID12 or variants based on all variable domains, an exceptionally advantageous feature combination, which is summarized as follows: recombinant human High affinity binding to CD70, strong binding to CD70 at the cell surface, specific cell lines expressing CD70, particularly in "low copy number" cancer cell lines binding to CD70 expressed in the vascular lines and cancer isolated from patient samples cells (CLL), strong blocking of CD70/CD27 interaction strong effector function - particularly in the form of a chimera with human IgG1 constant regions When expressed, and especially when expressed as a nonfucosylated IgG1, al cheeked (Rhesus) macaque and cynomolgus monkey with CD70 homologues in primate species cross-reactivity, which makes it possible to carry out toxicology studies, is both natural (i.e. cell on the surface), both binding to heat-denatured CD70, in some cancer cell lines partial or low levels of internalization. All of these features in combination 41 Dl 2 and indeed other 27B3 variants and exhibit similar properties as described here other CD70 antibodies, CD70-associated diseases that specifically express CD70 attractive for therapeutic use in the treatment of cancers and immunological disorders makes it a candidate. shown as heavy chain variable is the presence of the CDR3 sequence.

Here, the same heavy chain as 27B3 or human germline-treated 27B3 variants Antibodies containing a combination of CDR7s or antigen-binding fragments thereof is explained. Antibody or antigen-binding fragment a heavy chain may contain the variable domain: variable heavy chain CDR3 SEQ ID. amino acid of NO:50 or a sequence variant thereof contains or consists of a sequence; variable heavy chain CDR2 SEQ ID. NO:27, SEQ ID. No:249 and their series comprises or consists of an amino acid sequence selected from the group consisting of variants; and variable heavy chain CDR] SEQ ID. NO:11, SEQ ID. No:248 and their series contains or consists of an amino acid sequence selected from the group consisting of variants, wherein sequence variants include one, two or three amino acid substitutions in said sequence (for example, conservative substitutions, humanizing substitutions or affinity variants).

Antigen or antigen binding fragment HCDR3, HCDR2 and HCDR1 coinbination It may contain a heavy chain variable domain selected from: (a) DlZl ID. NO:50lyi (DAGYSNHVPIFDS) or a sequence variant thereof or variable heavy chain CDR3 consisting of; SEQ ID. NO: or a string variant thereof variable heavy chain CDR2 comprising or consisting of; and SEQ ID. NO: 1”i (VYYMN) or a number of variants thereof, or consisting of variable heavy chain CDR1, wherein sequence variants have one, two or three amino acid substitutions in said sequence (for example, conservative substitutions, humanizing substitutions or affinity variants); (b) SEQ ID. NO: or a string variant thereof or variable heavy chain CDR3 consisting of; SEQ ID. No: or a string variant thereof variable heavy chain CDRZ containing or consisting of; and SEQ ID. NO:11SI (VYYMN) or a number of variants thereof, or consisting of variable heavy chain CDR1, wherein sequence variants have one, two or three amino acid substitutions in said sequence (for example, conservative substitutions, humanizing substitutions or affinity variants). (0) SEQ ID. NO: or a string variant thereof or variable heavy chain CDR3 consisting of; SEQ ID. NO:27” (DINNEGGTTYYADSVKG) or a series variant thereof variable heavy chain CDRZ containing or consisting of; and SEQ ID. NO: or a sequence variant thereof, or the resulting variable heavy chain CDR1, wherein sequence variants have one, two or three amino acid substitutions in said sequence (for example, conservative substitutions, humanizing substitutions or affinity variants).

A light chain whose antibody or antigen-binding fragment matches the heavy chain variable domain It also contains the variable domain (VL). In preferred light chain variable domains, the variable light chain CDR3 SEQ ID. NO: 160.1 or a series variant thereof, or variable light chain CDRZ SEQ ID. N02] 19, SEQ ID. NO: 11 and the sequence of the mentioned sequences comprises or consists of an amino acid sequence selected from the group consisting of variants; and variable light chain CDR1 SEQ ID. NO:87, SEQ ID. NO:250, SEQ ID. No:251, SERIAL ID.

NO:252, SEQ ID. No: 253 and from the group consisting of sequence variants of the mentioned sequences contains or consists of a selected amino acid sequence; wherein sequence variants include one, two or three amino acid substitutions in said sequences (for example, conservative substitutions, humanizing substitutions or affinity variants).

The antigen or antigen binding fragment may contain a light chain variable domain, where the combination of LCDR3, LCDR2 and LCDR1 is selected from the following: (a) SEQ ID. NO: or a sequence variant thereof, or variable light chain consisting of CDR3; SEQ ID. No: or a number of variants thereof, or the resulting variable light chain CDR2; and SEQ ID. NO: or a string variant thereof or the variable light chain CDR1 composed of them, wherein sequence variants have one, two or three amino acid substitutions in said sequence (For example, conservative substitutions, humanizing substitutions or affinity variants); (b) the variable light chain contains or consists of CDR3 SEQ ID. NO: variable light chain comprising or consisting of or a sequence variant thereof SEQ ID. NO: or a sequence variant thereof, or the resulting variable light chain CDR2; and SEQ ID. NO:87° (GLKSGSVTSTNFPT) or a series variant thereof, or variable light chain consisting of CDR1, wherein sequence variants have one, two or three amino acid substitutions in said sequence (for example, conservative substitutions, humanizing substitutions or affinity variants). Other light chain CDRs of human "gerrnline processed" variants of 27B37 combinations are given in Table ISA.

Most preferred of the CD70 antibody or antigen-binding fragment thereof arrangement of a heavy chain variable domain (VH) and a light chain variable domain (VL) contains 6 CDRs that form the binding site for human CD70. combination includes the following: variable heavy chain CDR3 SEQ ID. NO: contains or it consists of; variable heavy chain CDR2 SEQ ID. NO:27lyi (DINNEGGTTYYADSVKG) or consists of; variable heavy chain CDRl SEQ ID. NO2] includes or consists of 1 (VYYMN); variable light chain CDR3 SEQ ID. NO: includes or variable light chain CDR2 SEQ ID. NO: includes or occurs; and variable light chain CDR1 SEQ ID. NO: contains or it consists of. Other disclosed combinations for 6 CDRs are Table 14A of 27B3 (heavy chains) and Occurring in human germline-operated variants listed at 15A (light chains) The above preference based on 27B3 and its germline-processed variants In the foregoing embodiments, the antibody is preferably a human antibody, especially notably CH1 domain, hinge region, CHZ domain of human IgG1, IgG2, IgG3 or IgG4, and It contains the CH3 domain. The most preferred arrangement is a human lgGl ”din Even more antibody-dependent cellular cytotoxicity (ADCC) of preferred human IgG1, complement-dependent cytotoxicity (CDC) or antibody-dependent cellular phagocytosis (ADCP) It is designed to maximize the effector function in excess. Especially preferred a non-fucosylated human IgG1, eg PotelligentTM technology from BioWa Inc. It is an unfucosylated IgG17 produced using

The human ship line of 27B3 and the Fab named 27B3, which are also described here, were processed. CD70 antibodies exhibiting high affinity binding to human CD70 based on variants of and SEQ ID. At least 90%, 95%, with one of the NO:275 amino acid sequences and said sequences, containing a heavy chain variable domain with an amino acid sequence and optionally SEQUENCE ID. No:276 and DIZI ID. No: 277 with one of the amino acid sequences and the aforementioned sequences. isolate containing a light chain variable domain having an amino acid sequence selected from the group Contains antibodies or antigen-binding fragments thereof.

The above listed VH and VL domain sequences of VH domains and VL domains Even if all possible matches selected from the groups are possible, some VH and VL combinations are particularly preferred; they have high affinity for human CD70. are "natural" combinations within a single Fab that exhibit binding. Table 14B of 27B3 and In the case of the germline-treated variants mentioned in 15B, the original It may be preferable to preserve the VH/VL match.

Thus, preferred CD70 antibodies exhibit high affinity CD70 binding. or their antigen-binding fragments a heavy chain variable domain and a light chain containing a combination of variable domains, wherein the combination is is as follows: (i) SEQ ID. a heavy chain variable domain containing the amino acid sequence of No:223, and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO:241; It also exhibits high affinity CD70 binding and is a heavy chain variable domain and CD70 antibodies containing a combination of light chain variable domains or their Antigen-binding fragments are also described, wherein the combination is one of the following: selected from the group consisting of: (ii) SEQ ID. a heavy chain variable domain containing the amino acid sequence of NO:178, and SEQ ID. a light chain variable domain containing the amino acid sequence of NO: 190; (iii) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:212 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 230; (iv) SEQ ID. No:213? A heavy chain variable domain containing the amino acid sequence of and SEQ ID. a light chain variable domain containing the amino acid sequence of NO: 23l; (v) SEQ ID. a heavy chain variable domain containing the amino acid sequence of NO:2147, and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 232; (vi) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:215 and SEQ ID. a light chain variable domain containing the amino acid sequence of NO: 235; (vii) SEQ ID. A heavy chain variable domain10 containing the amino acid sequence of NO:216 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 234.6; (Viii) SEQ ID. A heavy chain variable containing the amino acid sequence of NO:217 domain and SEQ ID. A light chain variable containing the amino acid sequence of NO: 235 domain; (ix) SEQ ID. No:218? A heavy chain variable domain containing the amino acid sequence of and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 236; (x) SEQ ID. a heavy chain variable domain containing the amino acid sequence of NO:219 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 237; (xi) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:220 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 238; (xii) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:22] and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 23921; (xiii) SEQ ID. A heavy chain variable containing the amino acid sequence of NO:222 domain and SEQ ID. A light chain variable containing the amino acid sequence of NO: 24021 domain; (xiv) SEQ ID. A heavy chain variable domain containing the amino acid sequence of #224 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 242; (xv) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:225 and SEQ ID. a light chain variable domain containing the amino acid sequence of NO: 243; (xvi) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:226 and SEQ ID. a light chain variable domain containing the amino acid sequence of NO: 244; (xvii) SEQ ID. A heavy chain variable containing the amino acid sequence of NO:227 domain and SEQ ID. A light chain variable containing the amino acid sequence of NO: 245 domain; (xviii) SEQ ID. A heavy chain variable containing the amino acid sequence of NO:228 domain and SEQ ID. A light chain variable containing the amino acid sequence of NO: 2463 domain; (xix) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:229 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 247; (xx) SEQ ID. A heavy chain variable domain10 containing the amino acid sequence of NO:223 and SEQ ID. a light chain variable domain comprising the amino acid sequence of NO: 244.6; (xxi) SEQ ID. A heavy chain variable domain containing the amino acid sequence of NO:223 and SEQ ID. A light chain variable domain containing the amino acid sequence of NO: 245.

For each specific VH/VL combination listed above, with the VH domain sequence mentioned It is also possible to combine it with a VL domain with a sequence of are included.

The most preferred embodiment is the human gene line engineered variant 41Dl2. a CD70 antibody based on the natural VH/VL combination or its antigen-binding is the fragment. So, here is the SEQ ID. From the amino acid sequence shown as NO:223, gerrnline-processed variants and affinity variants thereof, and at least a heavy chain variable domain (VH) containing or consisting of the amino acid sequence and SEQUENCE ID. From the amino acid sequence shown as NO:24l, its germline processing was done containing an amino acid sequence selected from the group consisting of identical amino acid sequences, or an antibody or its antigen comprising a light chain variable domain (VL) consisting of The linker fragment is also provided.

SEQ ID of the VH domain amino acid sequence. More than 100% with the sequence shown as NO: 223 Arrangements in which it exhibits little sequence identity, however, the amino acid sequence within the framework regions SEQ ID displaying variations. No:223, to HCDR] , HCDR2 and HCDR3 (respectively SEQ ID: N02] 1, 27 and 50) may contain identical heavy chain CDRs. Likewise, VL SEQ 1D of the domain amino acid sequence. Less than 100% with the sequence shown as NO: 241 Arrangements in which it exhibits sequence identity are still amino acid sequence within framework regions. SEQ ID displaying variations. NO:241 to LCDR1, LCDR2 and LCDR3 (respectively They may contain heavy chain CDRs identical to SEQ ID: Nos: 250, 116 and 160). Based on the VH and VL domains of 41D12 or variants thereof, the aforementioned in preferred embodiments, the antibody preferably refers to a human antibody, particularly CH] domain of human IgG1, IgGZ, IgG3 or IgG4, hinge region, CH2 if necessary domain and the CH3 domain. The most preferred embodiment is a human IgG1, More one or more of the ADCC, CDC, or ADCP of the more preferred human IgG1 It is designed to maximize the effector function in excess. Especially preferred from fucosyl, preferably prepared using the PotelligentTM expression system. a purified human is lgGl”.

Also SERIAL ID. From the amino acid sequence denoted No:225, its germline process made variants and affinity variants with at least 90%, 95%, 97%, 98% or containing an amino acid sequence selected from the group consisting of 99% identical amino acid sequences or consisting of a heavy chain variable domain (VH) and SEQ ID. Denoted by No:243 amino acid sequence, its germline engineered variants, and affinity A light compound containing or consisting of an amino acid sequence selected from the group consisting of An antibody or antigen-binding fragment thereof containing a chain variable domain (VL) is explained; or Also SERIAL ID. From the amino acid sequence denoted NO:226, its ship line processing made variants and affinity variants with at least 90%, 95%, 97%, 98% or containing an amino acid sequence selected from the group consisting of 99% identical amino acid sequences or consisting of a heavy chain variable domain (VH) and SEQ ID. Shown with No:244 amino acid sequence, its germline-processed variants, and affinity A light compound containing or consisting of an amino acid sequence selected from the group consisting of An antibody or antigen-binding fragment thereof containing a chain variable domain (VL) is explained.

Considerations/Characteristics of CD70 Antibodies In the above-mentioned aspects and embodiments, CD70 antibodies or their antigen each of the linker fragments has one or more of the following features or aspects may exhibit more or a combination of these: The antibody or antigen-binding fragment is human when tested as a Fab fragment. can bind with high affinity.

The antibody or antigen-binding fragment can bind to human CD70 with high affinity and and inhibit the interaction between CD27. Alternatively, antibody or antigen binding The fragment can bind to human CD70 but inhibit the interaction between CD70 and CD27. it may not.

The antibody or antigen-binding fragment is human on the surface of CD70-expressing cells. CD703e can bind with high affinity.

The antibody or antigen-binding fragment is human on the surface of CD70-expressing cells. It can bind to CD70 and be slowly or only partially intemalized. find it An important aspect is that CD70 antibodies include many CD70-expressing cancer cell lines. In fact, it is underestimated in many CD70-expressing cell lines. is the observation. This observation suggests that CD70 antibodies are associated with renal cell carcinoma cell lines. with previously published reports that they are rapidly internalized following attachment. has direct consequences. Following binding of CD70 antibodies to cancer cells The observation that they are very little intemalized is that many CD70-expressing cancers and indeed Therapeutic strategies for the treatment of CD70-associated immunological diseases antibody to a therapeutic agent, for example a cytotoxic or cytostatic drug fragment specifically from ADCC, CDC, or ADCP, but not to the use of immunoconjugates to which it binds. extraordinary to CD70 with antibody effector function in any or more strongly concluded that it should be based on high affinity binding. supports.

Amino acid sequence of antibody or antigen-binding fragment human CD70 HIQVTLAICSS (SEQ ID No:342) can be connected in; Antibody or antigen-binding fragment with monkey-derived CD70, specifically rhesus macaque (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus) CD70 may exhibit cross-reactivity with its homologues.

The antibody or antigen-binding fragment can be both native to human CD70 (e.g. a cell, For example, a cell line or a CD70-expressing cell isolated from a human patient. CD70 expressed on the surface of the cell), as well as heat-denatured recombinant human Can be connected to CD70.

In systems of antibody or antigen binding fragment recombinant antibody expression, for therapeutic antibody products, for example in the CHKl SV cell line (BioWa/Lonza,ya proprietary) Very high production yields (>4 g/L) compared to the old average of 1-2 g/L can provide, which leads to a significant reduction in production costs.10 The antibody or antigen-binding fragment should be stored under 37°C and freeze-dried. it can be quite stable in dissolving cycles, which is an important cost-reducing factor.

Antibody to antibodies to cells expressing the human CD70 protein on the cell surface cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent one or more effector functions selected from attached cell-mediated phagocytosis (ADCP) can display.

Antibody to cells expressing CD70, for example cancer cells or other malignant may exhibit ADCC to cells or to cells.

The antibody is combined with a reference antibody, which is an equivalent antibody containing a native human FC domain. can exhibit enhanced ADCC function in comparison. a non-irritating In one embodiment, the ADCC function is combined with a reference antibody containing a natural human Fc domain. may be at least ten times stronger in comparison. "Equivalent" in this context, strengthened ADCC Except for modifications made to potentiate the ADCC of a functional antibody Antigen binding specificity substantially identical to the reference antibody (of native human F c*) It can be considered in the sense that it shows and/or shares an identical amino acid sequence.

Conjugation of antibody or antigen-binding fragment to a cytotoxic or cytostatic agent It can inhibit tumor growth in an in vivo tumor xenograft model without Restrictive In a non-embodiment, inhibition of tumor growth function was by reference antibody SGN70. It can increase at least 10 times when compared.

Apoptosis in cells expressing the antibody or antigen-binding fragment CD70 can induce.

The antibody is hinged to a human IgGs, most preferably human IgG1, IgG2, IgG3, or IgG49e. region, the CH2 domain, and the CH3 domain.

Modifications in the antibody Fc region, such as antibody as described elsewhere herein may contain modifications that increase effector function. Specifically, the antibody is a nonfucosylated IgG it could be.

In other aspects, the invention includes the CD70 antibodies listed above and their antigen polynucleotide molecules encoding linker fragments, additionally containing polynucleotides expression vectors, host cells containing the vectors, and recombinant CD70 antibodies Methods for its expression/production are also provided.

In yet another aspect, any of the CD70 antibodies described above in the invention10 a pharmaceutical containing one and a pharmaceutically acceptable carrier or excipient composition is provided.

Another aspect of the invention is the above-listed aspects, particularly in the treatment of cancer. Relates to medical treatment modalities using CD70 antibodies.

Brief Description of Figures The invention is further elaborated by reference to the following experimental examples and the accompanying Figures. it will be understood: Figure 1: of lainae infused with 786-0 cells (top) and Raji cells (bottom) Immune response obtained in ELISA on recombinant CD70 is shown.

Figure 2: By ELISA with CD70 Fabs from llama and reference CD70 Fabs Inhibition of measured CD27 binding to CD70 is shown.

Figure 3: in a binding ELISA (black) or an inhibition ELISA (white) is a graphical representation of the signal for the F abs obtained from the llama under test.

Figure 4: ELISA with chimeric llama-human CD70 mAbs and reference CD70 mAbs of CD27 as measured by human CD70° (A&B') or rhesus monkey CD70° (C) inhibition of binding is shown.

Figure 5: 786-0 of chimeric llama-human CD70 mAb71s as evidenced by FACS analysis cells (A) or MHH-PREB-1 cells (B).

Figure 6: CD70° specificity in a Raj i cell-based co-culture potency analysis Inhibition of chimeric llama-human mAbs is shown.

Figure 7: Analysis of standard Cr51-release ADCC performed in 786-0 cells. results are shown.

Figure 8: CDC performed on U266 cells in the presence of 9% human serum The results of the analysis are shown.

Figure 9: Chimeric llama-human in an ADCP analysis performed on 786-0 cells The efficacy of CD70 mAbs is demonstrated.

Figure 10: Differential as a function of time in 786-0 cells in two independent experiments for chimeric llama-human CD70 mAbs evaluated as MFI OUTSIDE antibody internalization is shown.10 Figure 1 1: chimeric lama-human CD70 mAb 4lD12, isotype in a Raji Xenograft model The survival of mice after treatment with control and Fc-dead control is shown.

Figure 12: Amino acid sequences of VH3-38 and VL8-6l human germline gene segments, respectively Alignments of the VH and VL amino acid sequences of clone 27B3 with their sequences is shown.

Figure 13: After 5 weeks of incubation at 37°C, the germline procedure was performed. Gel filtration analysis of samples for 27B3 mAb variants is shown.

Figure 14: taken at various time points after incubation at various temperatures using Biacore for vessel line-treated CD70 mAb samples The potency of the measured CD70 binding is shown.

Figure 15: CD70 mAbs on CD70-expressing cancer cell lines binding affinity is shown.

Figure 16: CD70 mAbWeri in CD70-expressing CLL patient cells affinity is shown.

Figure 17: Lysis of SU-DHL-6 cells bound with CD70 mAbs is shown.

Figure 18: Binding of CD27 to CD70 measured in ELISA with CD70 mAbs inhibition is shown.

Figure 19: an alignment of CD70 sequences from different species is shown. Binding affinity for cynomolgus monkey HSC-F cells is shown.

Figure 21: Human, rhesus monkey CD27 identified in ELISAS with CD70 mAbs and inhibition of its binding to cynomolgus monkey CD70.

Figure 22: Denatured recombinant CD70 of CD70 mAbs assessed by ELISA binding is shown.

Figure 23: CD70 chimeric sequences used for epitope mapping are shown.

Figure 24: CD70 mAbs as a function of time in 786-0 cells antibody internalization is shown.

Definitions term two heavy and two light two, whether or not it has any specific immunoreactivity contains a polypeptide having a combination of chain. "Antibodies" to a related antigen groups with known significant specific immunoreactive activity for (e.g. human CD70) corresponds to. The term "CD70 antibodies" herein refers to the human CD70 protein immunological used to correspond to antibodies that exhibit specificity. here somewhere else crosses with strains with CD70 homologs "specificity" for human CD70, as described does not exclude the reaction. An inter-chain link between antibodies and immunoglobulins It includes light and heavy chains with or without covalent bonding. in vertebrate systems basic immunoglobulin structures are relatively well known.

The general term "immunoglobulin" is five different antibodies that can be biochemically differentiated. contains the class. All five classes of antibodies are within the scope of the present invention. The disclosure is generally directed to the IgG class of immunoglobulin molecules. pertaining to IgGly In general, immunoglobulins are two identical light molecules with a molecular weight of approximately 23,000 Daltons. associated with disulfide bonds in a "Y" configuration, where the light chains are It supports heavy chains starting at the mouth and continuing through the variable region.

The light chains of an antibody are classified as kappa or lambda (K, l). Every heavy chain class either kappa or lambda may be linked by a light chain. In general, light and The heavy chains are covalently linked together and the "tails" of the two heavy chains by covalent disulfide linkages or immunoglobulins hybridomas, B cells or covalent when created with genetically engineered host cells connected with non-existent connections. In the heavy chain, the amino acid sequences are forked in the Y configuration. extends from an N terminal at the ends of each chain to the C terminal at the bottom of each chain. technical expert those of heavy chains are gamma, mu, alpha, delta or epsilon, (y, u, oi, ö, 8) classified and that there are some subclasses (eg, 71 - y 4) among them. will know. It is this that determines the antibody "class"1 in the form of IgG, IgM, IgA, IgD or IgE, respectively. is the structure of the chain. Immunoglobulin subclasses (isotypes), eg IgG1, IgG2, IgG3, are known to provide. Modified variants of each of these classes and isotypes can be easily distinguished by experts, taking into account the present description, and therefore falls within the scope of the present invention.10 As noted above, the variable region of an antibody is the epitopes of the antibody in antigens. allows it to selectively recognize and specifically bind to them. That is, an antibody The VL domain and the VH domain combine to define a three-dimensional antigen-binding domain. creates the variable region. This quadrilateral antibody structure is located at the end of each arm of the shin. forms the antigen binding site. More specifically, the antigen binding site VH and three complementarity determining regions (CDRs) in each of the VL chains. is defined. or the terms "CD70 antigen" or "CD70" may be used interchangeably and It corresponds to a member of the TNF ligand family, which is a ligand for TNFRSF27/CD27. "Human The terms "CD70 protein" or "human CD70 antigen" or "human CD70" are interchangeable may be used, corresponds specifically to the human homolog and naturally natural human expressed in the body and/or on the surface of cultured human cell lines It contains the CD70 protein as well as recombinant forms and fragments thereof. Human Specific examples of CD70 Accession No. NP_0011243 NCBl Reference Sequence shown under contains the polypeptide having the amino acid sequence or its extracellular domain. a region responsible for selective binding to a target antigen of interest (eg human CD70). includes. Binding domains contain at least one binding domain. Example binding domains contains the antibody variable domain. Antibody molecules of the invention have a single binding site or may contain multiple (for example, two, three, or four) binding sites. a CD70 antibody or antigen-binding fragment thereof) "obtained" refers to the polypeptide corresponds to its origin. In one embodiment, derived from a particular starter polypeptide the polypeptide or amino acid sequence is a CDR sequence or a sequence associated with it. A in one embodiment, the amino acid sequence from a particular starter polypeptide is not contiguous. For example, in one embodiment, one, two, three, four, five, or six CDRs are obtained from a starter antibody. is done. In one embodiment, derived from a particular starting polypeptide or amino acid sequence. polypeptide or amino acid sequence with that of the start sequence or a portion thereof It has an identical amino acid sequence, where the fraction consists of at least 3-5 amino acids, 5-10 acid or originating from the initial sequence, a person of general knowledge in the art can be determined by In one embodiment, one or more 10 from the starter antibody variant CDR sequences, eg affinity variants, are produced by replacing excess CDR sequences wherein variant CDR sequences retain CD70 binding activity. antibody molecules elicited by active immunization of a camel with the CD70 antigen. framework amino acid sequences and/or CDR from a camelid conventional antibody Contains amino acid sequences. However, amino acid sequences from camelid containing CD70 antibodies from a human amino acid sequence (ie, a human antibody) or camel will contain framework and/or constant region sequences from another mammalian species that is not can be designed in . For example, a human or non-human antibody to said CD70 antibodies The primate framework region, the heavy chain portion, and/or the hinge portion may be included. A in the embodiment, one or more non-camelidae amino acids are "derived from camelids" may be present in the framework region of a CD70 antibody, for example, a camelid framework amino the presence of the corresponding human or non-human primate amino acid residue in the acid sequence. It may contain one or more amino acid mutations. In addition, it is obtained from camelid family the VH and VL domains or humanized variants thereof by linking to its fixed domains, as described in detail elsewhere here. chimeric molecule can be obtained. a residue in which the residue is replaced by an amino acid residue with a similar side chain. is a substitution. Families of amino acid residues with similar side chains have been identified in the art. These are basic side Chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (for example, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine) includes. Thus, a non-essential amino acid in an immunoglobulin polypeptide residue can be replaced by another amino acid residue from the same side chain family. Another in one embodiment, the sequence and/or composition of side chain family members of an amino acid sequence can be replaced in a structurally similar order, which differs in terms of It contains amino acid sequences derived from the constant domains of the immunoglobulin heavy chain.

A polypeptide containing a heavy chain portion is a CH1 domain, a hinge (eg, upper, middle and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or one of these variant or fragment thereof. In one embodiment, an antibody of the invention or The antigen-binding fragment represents the Fe portion of an immunoglobulin heavy chain (for example, a hinge fraction may contain a CH2 domain and a CH3 domain). In another embodiment, an inventive The antibody or antigen-binding fragment contains at least a portion of a constant domain (for example, a CH2 may not contain all or part of the domain. In some embodiments, fixed domains at least one, and preferably all, is derived from a human immunoglobulin heavy chain. For example, In a preferred embodiment, the heavy chain portion includes a fully human hinge domain.

In other preferred embodiments, the heavy chain portion is a fully human PC portion (eg, hinge of a human immunoglobulin, CH2 and CH3 domain sequences).

In some embodiments, the constitutive constant domains of the heavy chain portion are different immunoglobulins. is one of the molecules. For example, the heavy chain portion of a polypeptide is derived from an IgG1 molecule. a CH2 domain derived from an IgG3 or IgG4 inolecule and a hinge region may contain. In other embodiments, the constant domains belong to different immunoglobulin molecules. are chimeric domains containing fractions. For example, a hinge is a first from an IgG1 molecule. part and a second part of an IgG3 or IgG4 molecule. as stated above For example, one of ordinary skill in the art can determine the constant domains of the heavy chain portion of the amino acid differs from naturally occurring (wild-type) immunoglobulin molecule in sequence will know that they can be modified to show That is, belonging to the invention described here polypeptides in one or more of the heavy chain constant domains (CH1, hinge, CH2 or CH3) and/or changes in the light chain constant region domain (CL), or may contain modifications. Exemplary modifications are in one or more domains. includes additions, deletions or substitutions in excess amino acids. It contains a first amino acid sequence attached to the sequence. Amino acid sequences are normally They may be present in separate proteins assembled into a polypeptide or normally in the same may be present in the protein but has been put in a new arrangement in the fusion polypeptide they may be. A chimeric protein can be obtained, for example, by chemical synthesis or where peptide regions are desired. can be created by creating an encoded polynucleotide in the relationship and applying translation. Exemplary chimeric CD70 antibodies are the result of a human antibody, such as human IgG1, IgG2, IgG3, or or humanized variants thereof. The terms are used interchangeably herein and have equivalent meanings.10 vary widely in terms of the extent to which each specific antibody is directed against its target antigen. corresponds to its use in binding and specificity. With this, variability is not evenly distributed in the variable domains of antibodies. Antigen binding "extremely variable loops" in each of the VL domain and the VH domain that make up the It is concentrated in three parts called First, second, and third excess of the VLambda light chain domain The variable loop is referred to here as L1(7\.), L2(›\.) and L3(›\.), and the residue in the VL domain is 24-33 ,ü 90-96”y1 (L30t consisting of 5 residues)) (Morea et al., Methods residue L3(K)) (Morea et al., Methods 20:267-279). (2000)). The first, second, and third hypervariable loops of the VH domain are here H1, HZ, and H3. or H2) consisting of 4 residues and 91-1057i (H3, highly variable in length) Unless otherwise noted, the terms L1, L2, and L3 represent the first, second, and second half of a VL domain, respectively. corresponds to the third hypervariable loops and each of the Vkappa and Vlambda isotypes includes extremely variable stitches derived from both. The terms HI, H2, and H3 are VH, respectively. corresponds to the first, second, and third hypervariable loops of the domain and y, 8, 8, (1 or p hypervariable loops from any of the known heavy chain isotypes, including The hypervariable loops L1, L2, L3, H1, H2, and H3 are each one as described below. The definition of complementarity determining regions (CDRs) depends on sequence variability. (Kabat et al., Sequences of Proteins of Immunological are not completely synonymous due to the fact that HVs and CDRs are limited by some VH and VL may differ in their domains.

CDRs of the VL and VH domains typically contain the following amino acids may be specified: residue in the light chain variable domain and 89-10 97 (CDRLS) and residue in the heavy chain variable domain , ; (Kabat et al., Sequences of Proteins of Immunological Thus, HVs can be found within the corresponding CDR°s, where VH and VL references to "overvariable loops" of domains unless otherwise noted should also be interpreted to include the corresponding CDRs and vice versa.

The more highly conserved portions of the variable domains are described below. It is called the frame region (FR). Variable of natural heavy and light chains each of the domains contains four FRs (FR1, FR2, FR3, and FR4 respectively), which are large It takes a configuration of [3 leaves] combined with three highly variable loops. in every chain The overly variable loops are held very close to each other by FRs and the excess of the other chain contribute to the formation of the antigen-binding domain of antibodies with variable loops they are found. Structural analysis of antibodies by complementarity determining regions. revealed the relationship between the sequence and shape of the created binding site (Chothia et al. 182 (1990)). Despite their high sequence variability, five of the six loops are main chains. From its conformations it acquires only a small repertoire of so-called 'canonical structures'. This conformations are primarily related to the length of the loops and secondly to the loops and frame. in some residues, they may be packaged, hydrogenated, or important residues that determine the conformation with their ability to take the chain conformations. are identified by their presence. The term is adjacent to the variable region of both heavy and light chain polypeptides. means non-antigen combining domains. These special regions are described in Kabat et al., J. Biol. contains overlapping or subset amino acid residues when compared with each other.

Amino encompassing CDRs as defined in each of the above references acid residues are indicated for comparison. Preferably, the term "CDR" It is a CDR as defined by Kabat based on comparisons.

Table 1: CDR Definitions CDR Definitions Kabat] Chothia2 MacCallum3 lResidue numbering is according to the nomenclature of Kabat et al. (see above) 2Residue nuinalandirm according to the nomenclature of Chothia et al. (see above) 3Residue enumeration refers to the nomenclature of MacCallum et al. (see above). is relative that are part of the variable region but not of CDRs (for example, for CDRs) Using the definition Kabat) contains amino acid residues. Therefore, a variable region framework is about 100-120 amino acids in length, but only CDRs Contains essential amino acids. A heavy chain as defined by Kabat and others In the specific example for the variable domain and CDRs, the framework region 1 amino acids 1-30 corresponds to the spanning variable region domain; framework region 2 amino acids 36-49°u corresponds to the spanning variable region domain; framework region 3 amino acids 66-94 corresponds to the encompassing variable region domain, and the frame region 4 is variable from 103° corresponds to the variable region domain, which includes amino acids up to the end of the region.

The light chain framework regions are likewise associated with each of the light chain variable region CDRs. are separated. Likewise, for the CDR7s of Chothia et al. or McCallum et al. When the definition is used, the frame region limits are as described above for the corresponding CDR. separated by terminals. In preferred embodiments, the CDRs are defined by Kabat. is in the form.

In naturally occurring antibodies, the six CDRs found in each monomeric antibody are short, contiguous non-aino acid sequences, which are three-dimensional in an antibody aqueous medium. specifically to form the antigen binding site while taking its configuration. is located. The rest of the heavy and light variable domains are in terms of amino acid sequence. show less intermolecular variability and are expressed as framework regions. Framework regions largely take a [3-leaf conformation, and CDRs [3-leaf it forms loops that join its structure and in some cases form part of it.

Thus, these framework regions are linked by interchain, non-covalent interactions of the six CDRs. It functions as a scaffolding that ensures that it is positioned in the correct orientation. positioned Antigen binding site formed by CDRs to epitope in immunoreactive antigen defines a complementary surface. The immunoreactive antigen of this complementary surface antibody promotes non-covalent binding to its epitope. Location of CDRs general information in the art The owner can be easily identified by a person. It contains the part of the molecule that joins the CH1 domain to the CH2 domain. This is the hinge area contains approximately 25 residues and is flexible, thus two N-terminal antigen binders It allows the region to move independently. Hinge regions upper, middle and lower hinge domain may contain one of the hinge region sequences.

Table 2: Human Hinge Sequences lgG Top Hinge Middle Hinge Bottom Hinge LGG42 ERK CCVECPPPCP APPVAGP when numbering schemes are used (residues 244 to 360, Kabat numbering system; and residue 231-340, EU numbering system, Kabat EA et al., Sequences of Proteins of Immunological Interests. Bethesda, US Department of Health and Human Services, NIH. 1991) residue from about 244 of a heavy chain molecule, for example an antibody It includes the part extending to 360.1na. CH2 domain is not closely matched to another domain it is special in its own way. Rather, it exists between the two CH2 domains of an intact natural IgG molecule. There are two N-linked branched carbohydrate chains. CH210 of the IgG molecule of the CH3 domain It is also quite good that it extends from the C domain to the C terminal and contains approximately 108 residues. is documented. a fragment containing fewer amino acid residues than the antibody or antibody chain, or corresponds to the part. The term "antigen-binding fragment" refers to an immunoglobulin or with the antibody that binds the antigen or is intact (ie, with the intact antibody from which it has been obtained) a polypeptide that competes for binding to the antigen (ie, specific binding to human CD70) corresponds to the fragment. As used herein, the "fragment" of an antibody molecule the term antigen-binding fragments of antibodies, for example an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single-chain antibody (SCFV) includes an F(ab')2 fragment, a Fab fragment, an Fd fragment, an FV fragment, and a single contains a domain antibody fragment (DAb). Fragments, for example, intact or whole by chemical or enzymatic treatment of the antibody or antibody chain, or can be obtained by recombinant means. corresponds to the number of binding sites. Each target binding site specifically binds to the target molecule or to a specific site in a target inolecule. More than one polypeptide contains a target binding site, each target binding site is specifically the same or different binds molecules (for example, to different ligands or to different antigens, or to different antigens in the same antigen) may bind to epitopes). These binding molecules are specific for a human CD7O molecule. has at least one attachment area. corresponding to its capacity to be immunoreactive. A polypeptide can be monospecific and may contain one or more binding sites that specifically bind a target, or The polypeptide can be multispecific and specifically binds two or more of the same or different targets. may contain more attachment areas. In one embodiment, an antibody of the invention has more than one is specific to the target. For example, in one embodiment, a multispecific binding molecule of the invention It binds to CD70 and a second molecule expressed in a tumor cell. Tumor sample containing antigen-binding domains that bind to antigens expressed on cells antibodies are known in the art and one or more of the CDRs of such antibodies belong to the invention. they can be incorporated into an antibody. It contains polypeptides containing an amino acid sequence that has not been formed as a component. For example, naturally Ungenerated polypeptides are modified forms of naturally occurring polypeptides (for example, add-on, containing a mutation, such as a substitution or deletion) or in a linear sequence of amino acids a second amino acid sequence (naturally occurring or an attached first amino acid sequence (which may not have occurred naturally or may not have occurred). manipulation of polypeptide molecules by synthetic means (e.g. recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or with some combination of techniques). Preferably, antibodies of the invention are designed, e.g. humanized and/or chimeric antibodies and one or more traits, e.g. for enhancement of antigen binding, stability/half-life or effector function designed antibodies. modified synthetic forms so that they would not have occurred naturally, for example at least two antibodies that contain a heavy chain portion but not two full heavy chains (for example, the domain deleted antibodies or mini-antibodies); antibodies to two or more different antigens or multispecific forms that have been modified to bind to different epitopes on a single antigen (eg, bispecific, trispecific, etc.); heavy chain molecules associated with scFv molecules and includes the like. ScFv molecules are known in the art and, for example, in US Patent No. 5,892,019. are described in the document. In addition, the term "modified antibody" refers to the multivalent forms (for example, trivalent, four-valent, which binds to three or more copies of the same antigen) valuable, Etc. contain antibodies). In another embodiment, a modified antibody of the invention is a fusion protein that contains at least one heavy chain segment that is not a CH2 domain and a polypeptide containing the binding portion of a member of the receptor ligand pair. contains the attachment area.

The term "modified antibody" herein refers to amino acid sequence variants of a CD70 antibody. can also be used as a counterpart. One of ordinary skill in the art is able to identify a CD70 antibody, amino acid sequence compared to the CD70 antibody from which it is derived modified to produce a variant CD70 antibody that varies in terms of will know it can be done. For example, preservatives on "non-essential" amino acid residues nucleotide or amino acid substitutions that lead to substitutions or changes (for example, in CDR and/or frame remnants). Amino acid substitutions a10 or more amino acids with a naturally occurring or non-naturally occurring amino acid may include replacement. The term "substitutions" refers to a CD70 antibody (for example, a camelid CD70 antibody) is an amino acid residue present in a particular subject in the VH or VL domain. with an amino acid residue occurring at an equivalent position in the reference human VH or VL domain corresponds to amino acid substitutions in which it is modified. Reference human VH or VL The domain may be a VH or VL domain encoded by the human germline. humanizing substitutions in framework regions of a CD70 antibody described herein and/or It can be done on CDR7s. when compared to a reference CD70 antibody, one or more "humanizing" refers to a variant antibody containing a "substitution", wherein the reference antibody is a partial (for example, the VH domain and/or the VL domain, or parts thereof containing at least one CDR) has an amino acid derived from a non-human species and has "humanizing substitutions" occurs in the amino acid sequence derived from a non-human species. The term is used specifically to refer to "humanized variants", where CD70 antibody) is one or more present at a specific location(s) in the VH or VL domain. a reference human VH encoded by the human germline of further amino acid residues or its replacement by an amino acid residue occurring at an equivalent location in the VL domain gives the result. Typical case germline for a given "germline-processed variant" Substitution amino acid residues substituted for the engineered variant solely or predominantly It is taken from the VH or VL domain encoded by a single human germline. Here used as a substitute. One or more "humanizing substitutions" to a VH or VL domain from camelids (for example, from llamas) insertion of "humanized" VH or VL domain from camelid (llama) will result in the production of the "variant". If the substituted amino acid residues are predominantly or from the VH or VL domain sequence encoded by only a single human germline. then the result is that the VH or VL domain from camelids (llamas) is "human". may be a variant with germline processing.10 One or more changes in amino acid sequence compared to CD70 antibody corresponds to a variant antibody that exhibits exhibits an altered affinity for the human CD70 protein compared to the antibody. Typical As a result, affinity variants for human CD70 compared with the reference CD70 antibody. exhibit an altered affinity. Preferably with the affinity variant reference CD70 antibody It exhibits enhanced affinity for human CD70 in comparison. Further development for human CD70 A slower dissociation rate for a lower KD or human CD70 or a non-human CD70 may be evident by a differentiation in the cross-reactivity pattern with its homologues. affinity Amino acid variants typically found in CDR7s when compared to the reference CD70 antibody. exhibit one or more changes in the sequence. These substitutions are in CDR°s to a certain extent. a naturally occurring amino acid residue of the original amino acid located at with a different amino acid residue, which may be a non-forming amino acid residue may cause change. Amino acid substitutions may or may not be protective. may not be. an antibody containing the variable domain (VL), if the VH domains and the VL domains are addressed together at least 90% amino acids with the most closely matched human germline VH and VL sequences when taken If it exhibits sequence identity, it is considered to have high human homology. high man Antibodies with homology to human germline sequences % sufficiently high sequence identity antibodies containing the VH and VL domains of natural non-human antibodies exhibiting may contain, for example, the VH and VL domains of conventional camelid antibodies. antibodies, as well as any designed, especially humanized or Contains germline-treated variants as well as "fully human" antibodies.

In one embodiment, the antibody's high human homology VH domain F R1, F R2, F R3 and F R4 an amino acid of 80% or more with one or more human VH domains in the framework regions may exhibit acid sequence identity or sequence homology. Inventive in other embodiments between the VH domain of the polypeptide and the most closely matched human germline VH domain sequence. amino acid sequence identity or sequence homology 85% or greater, 90% or greater, In one embodiment, the antibody's high human homology, the VH domain, is the most closely matched human Compared to the VH sequence, the framework region is one or more along FR1, FR2, FR3, and FR4. may contain more (for example, 1 to 10) amino acid sequence mismatches.10 In another embodiment, the antibody's high human homology VL domain framework region 80% or more with one or more human VL domains throughout FR1, FR2, FR3, and FR4 may exhibit a large sequence identity or sequence homology. Inventive in other embodiments between the VL domain of the polypeptide and the most closely matched human germline VL domain sequence. amino acid sequence identity or sequence homology 85% or greater, 90% or greater, In one embodiment, the antibody's high human homology, the VL domain, is the most closely matched human Compared to the VL sequence, the framework region is one or more along FR1, FR2, FR3, and FR4. may contain more (eg 1 to 10) amino acid sequence mismatches.

Sequence identity between antibody with high human homology and human germline VH and VL canonical folds can be determined before analyzing the percentage of H] and Human germline with identical canonical fold combination for H2 or L] and L2 (and L3) Defines the family of cross-sections. Subsequently, the variable region of the antibody of interest human germline family member with a high degree of sequence homology selected for grading. Chothia of hypervariable loops L1, L2, L3, H1, and H2. Determination of canonical classes can be found at www.bi0inf.0rg.uk/abs/chothia.html.page can be achieved through publicly available bioinformatics tools. The output of the program is a data Shows the lock residue requirements in the file. In these data files, key residual locations shown with the amino acids allowed at each position. Variable region of the antibody of interest sequence is given as input and is first aligned with a consensus antibody sequence in Kabat. numbering scheme is assigned. Martin and Thomton in the analysis of canonical folds A group lock residue pattern obtained by the method is used.

Using the same canonical folding combination for H] and H2 or L1 and L2 (and L3) family member best matched in terms of sequence homology with known specific human germline V-section can be determined. VH and VL domain framework amino acid sequences of the antibody of interest and human germ Percentage of sequence identity among the corresponding sequences encoded by the line It can be calculated with bioinformatics tools, but it can actually be calculated by manual alignment of sequences. can be printed. Human immunoglobulin sequences, for example VBase (httpz//vbasemrc- cpe.cam.ac.uk/) or the Pluckthun/Honegger database (http://www.bioc.unizh.ch/antibody/Sequences/Germlines for various protein data identifiable from their base. VH or VL domains of an antibody of interest in human sequences A set of alignment algorithms for comparison with V regions, e.g. 10 Available on web pages such as WWW.expasy.ch/t001s/#align are also limited Manual alignment with the array group can also be performed. With frame region 1, 2 and 3 of each chain belong to families with the same canonical fold combinations and the highest degree of homology human germline light and heavy chain sequences are selected and compared to the variable region of interest; In addition, FR4 is compared to the human germline JH and JK or J L regions.

In the calculation of percent total sequence homology, FR1, FR2, and FR3 residues were calculated using human germ The closest matching sequence, which is a combination of identical canonical folds from the line family It should be known that it is examined with its use. Only residues different from the closest match or other members of the same family with the same combination of canonical folds are scored (Note - excluding differences encoded by the primer). However, for humanization purposes, the same canonical with members of other human germline families that do not have a combination of folds Remains of identical frame regions are "negative" according to the mandatory conditions described above. Despite their scoring, they can be considered "human". This assumption is about humanization. individually compared to the most closely matched human germline sequence and therefore The humanized molecule contains a combination of different FRs. Chothia numbering Using the IMGT numbering scheme, which is an adaptation of the the boundaries of the frame region can be determined (Lefranc et al., NAR 27: 209-212 (1999); imgt.cines.fr).

Antibodies with high human homology are either human or may include hypervariable loops or CDRs with human-like canonical folds. A the antibody with high human homology in the embodiment, both in the VH domain and in the VL to a non-human antibody with at least one hypervariable loop or CDR in its domain, derived from a VH or VL domain of a conventional antibody, for example, from the camelid species can be derived or derived, yet with a canonical folding structure occurring in human antibodies exhibits a substantially identical predictive or true canonical folding structure.

in both VH domains and VL domains encoded by the human germline The primary amino acid sequences of the highly variable loops found are by definition highly variable. However, all hypervariable loops except CDR H3 of the VH domain are only canonical. It is well known in the art that it takes several distinct structural conformations called folds. is based on the presence of so-called acid residues (Chothia et al., J. Mol. their canonical structure can be determined by structural analysis (eg X-ray crystallography), but canonical predicting the structure based on key amino acid residues that are characteristic of a particular structure possible (described in more detail below). Essentially, every canonical the specific pattern that determines the structure creates a 'signature', in this way a VH or VL domain in hypervariable loops of an unknown structure, the identification of the canonical structure is provided; therefore, canonical structures can be predicted based only on the primary amino acid sequence.

The excess of any given VH or VL sequence of an antibody with high human homology predicted canonical folding structures for variable loops WWW.bi0inf.0rg.uk/abs/ch0thia.html, WWW.biochem.ucl.ac.uk/~martin/antib0dies.html and On the web pages WWW.bioc.unizh.ch/antibody/Sequences/Germlines/Vbase_hVk.html can be analyzed using publicly available algorithms. These tools are queried VH or VL alignment of sequences with known canonical human VH or VL domain sequences. makes it possible and predicts a canonical structure for hypervariable loops of the queried array makes.

In the case of the VH domain, if at least the first of the following criteria, and preferably each if both are met, the H] and H2 loops are the canonical folding known to occur in human antibodies It can be scored as having a canonical folding structure that is "substantially identical" to its structure: 1. The closest match to the human canonical structural class, determined by the number of remains having an identical length. 2. Key amino acids described for the corresponding human H1 and H2 canonical structural classes at least 33% identity, preferably at least 50% identity with acid residues. (For the purposes of the above analysis, loops Hl and HZ are processed separately and each compared to the closest matching human canonical structural class) The above analysis revealed the canonical structure of the H1 and HZ loops of the antibody of interest. based on forecasting. The actual structures of the H1 and H2 loops in the antibody of interest, for example X if the thread is known from crystallography and if the loop length is differs from that of the matching human canonical structural class (typically ±1 or ±2 amino acid) but the actual structure of the H1 and HZ loops in the corresponding antibody is a human canonical if it matches the folding structure, then the H] and H2 loops in the corresponding antibody are also human10. "substantially identical" with a canonical folding structure known to occur in antibodies can be scored as having a canonical folding structure.

Human related to the first and second hypervariable loops (HI and H2) of the human VH domains Key amino acid residues found in canonical structural classes are described by Chothia et al., J. Mol. in Table 3 on page 802 of the reference to Chothia et al. Preferred amino acid in key areas related to H1 canonical structures in the line remains are listed in Table 4 on page 803, which is also specifically included in this text. preference in key areas of CDR H2 canonical structures found in the human germline. amino acid residues are listed.

In one embodiment, each of H1 and H2 in the VH domain of the antibody with high human homology both are largely identical, with a canonical folding structure occurring in human antibodies. exhibits a predicted or true canonical folding structure.

Antibodies with high human homology, hypervariable H] and HZ loops have at least one human an identity identical to a combination of canonical structures known to occur in the germline VH domain contains a VH domain formed by the combination of canonical folding structures. human germ In the VH domains encoded by the line actually canonical folding in H] and H2 It has been observed that only some combinations of the structures are formed. In an arrangement H1 and H2 in the VH domain of the antibody with high human homology to a non-human species, may be obtained from a VH domain of, for example, a camelid species, but still a human known to occur in the germline or somatically inuted VH domain an identical predicted or true canonical combination with canonical folding structures form a combination of folding structures. High in non-irritating arrangements H] and H2 in the VH domain of the antibody with human homology to a non-human species, e.g. and form one of 3-5 canonical fold combinations.

An antibody with high human homology high sequence identity/sequence homology with human VH A VH containing hypervariable loops exhibiting structural homology with human VH. may contain the domain.

present in H1 and H2 in the VI-I domain of an antibody with high human homology of the folds and their combination, the VH of the antibody with high human homology Total primers for the human VH germline sequence representing the closest match to the It may be advantageous if it is "correct" in terms of amino acid sequence identity. For example, if most if the close sequence match is with a human germline VH3 domain, then the natural sequence of H] and H2 a combination of canonical folds that also occur in a human VH3 domain may be advantageous. This is the case for higher human species derived from non-human species. antibodies with homology, for example from conventional camelid antibodies Antibodies containing the VH and VL domains, particularly humanized camelid VH and VL This can be particularly important in the case of antibodies containing .

Thus, in one embodiment, the VH domain of the CD70 antibody with high human homology 80% or more with a human VH domain in the FR1, FR2, FR3, and FR4 framework regions, may exhibit a sequence identity or sequence homology until or even 100% The H] and H2 in the antibody are derived from a non-human VH domain (for example, a camel species) but a canonical known to occur naturally in the same human VH domain a predicted or actual canonical fold structures that are the same as the fold combination creates the combination.

In other embodiments, L1 and L2 in the V1 domain of the antibody with high human homology each from a VL domain from a non-human species (for example, a VL from camelids) domain) and each with a canonical folding structure formed in human antibodies exhibits a predicted or true canonical folding structure that is largely identical.

Just as with VH domains, VL in both Vlambda and VKappa types The hypervariable loops of its domains have a limited number of conformation or canonical structures. This is partly in length, but also in some canonical positions for key amino acids. determined by the presence of residues.

In a related antibody with high human homology, a non-human species, for example a If the L1, L2 and L3 loops obtained from the VL domain of the Camelidae (Camelidae) species in human antibodies if at least the first, and preferably both, of the following criteria are met: A canonical fold that is "substantially identical" with a canonical fold structure known to occur It can be scored as having the structure: 1. An identical to the most closely matched human structural class, determined by the number of remains have length. 2. The corresponding human L1 and L2 from both the Vlambda and VKappa repertoires at least 3310% with key amino acid residues described for their canonical structural class Identity, preferably at least 50% identity. (For the purposes of the above analysis, loops L1 and L2 are processed separately and each compared to the closest matching human canonical structural class) The above analysis shows that the L1, L2, and L3 loops in the VL domain of the antibody of interest are canonical. based on the prediction of its structure. The actual structure of the L1, L2 and L3 loops, eg X ray is known based on crystallography, and if the loop length is the closest match differs from that of the human canonical structural class (typically ±1 or ±2 amino acids) but the actual structure of Camellia (Cameli'dae) loops is with a human canonical folding. matches, then the L1, L2 and L3 loops from the corresponding antibody are also human. "substantially identical" with a canonical folding structure known to occur in antibodies can be scored as having a canonical folding structure.

Human canonical structural framework for the CDRs of the human VLambda and VKappa domains. key amino acid residues found in the classes Morea et al., Methods, 20: 267-279 is explained. Also, the structural repertoire of the human VKappa domain has been described by Tomlinson et al. L1 and L2 in the VL domain of an antibody with a predicted or real one identical to a known combination of canonical folding structures can form a combination of canonical folding structures. In non-irritating arrangements an antibody with high human homology (for example, a VL domain from camelid or an antibody containing a humanized variant thereof) L1 in the VLambda domain and (Williams et al., J. Mol. Biol. 2642220 -32 (1996) and On the website http://www.bioc.uzh.ch/antibody/Sequences/Germlines/V Base_hVL.htm1 as shown). In non-limiting embodiments, L1 and L2 in the Vkappa domain are 2-1, It can form one of the 3-1, 4-1 and 6-1 canonical fold combinations (Tomlinson et al. httpz//Www.bioc.uzh.ch/antibody/Sequences/Geimlines/VBase_h on the VKhtml website as shown).

In another embodiment, L1, L2 in the VL domain of an antibody with high human homology and all of L3 may exhibit significant human artifacts. with high human homology the antibody's VL domain exhibits high sequence identity/sequence homology with human VL, and In addition, hypervariable loops in the VL domain exhibit structural homology with human VL. preferable.

In one embodiment, the VL domain of a CD70 antibody with high human homology is F R1, F R2 , 80% or more, 85% or more with a human VL domain in the FR3 and FR4 framework regions more, 90% or more, 95% or more, 97% or more up to 99% or even 100% can exhibit a series of identities, and in addition, excessively variable loop L1 and excessive variable loop L2 is a canonical known to occur naturally in the same human VL domain. a predicted or actual canonical fold structures that are the same as the fold combination can make a combination.

Undoubtedly, high sequence identity/sequence homology with human VH as well as human VH excess VH domains, which exhibit structural homology with variable loops, have a high sequence with human VL. Identity/sequence homology as well as structural homology with hypervariable loops of human VL with VL domains exhibiting human-encoded VH/VL in this way. VH/VL pairs (e.g., VH/VL pairs) showing maximum sequence homology and structural homology with camelid-derived VH/V1 pairs) antibodies with high human homology provision is foreseen.

As summarized above, the invention shows that high affinity binds, at least in part, to human CD707. relates to antibodies and their antigen-binding fragments. CD70 according to the invention The properties and characteristics of antibodies and antibody fragments are now in more detail. will be disclosed.

CD70 Binding and Affinity In some aspects, the antibodies and antigen-binding fragments thereof provided herein binds to human CD703 with high affinity. Antibodies that bind to human CD70 with high affinity or antigen-binding fragments thereof for human CD70 and more particularly for human CD70 for extracellular domain about IOnM or less or lnM or less or 0.1nM or less, or a binding affinity (KD) of 10pM or less.

CD70 antibody (or antigen-binding fragment) in a Fab form of the VH and VL domains less than 7 x 10'4s`l for human CD70 binding, if expressed, may include VH and VL domains, which exhibit a rate of dissociation. Typically the rate of departure is 0.4 X 10`4s`10 It will range from 1 to 4.8 x 1045'1. Attenuation of attachment (KD) and rate of dissociation (Koff) standard techniques well known to those skilled in the art, for example those described in the accompanying examples. can be measured using surface plasmon resonance (BIAcore). Briefly, 50 pl to be tested sample + 195 u] HBS-EP+ is diluted. Biacore analysis room temperature highly CD70 coated CMS chip It is performed using the protocol provided by the manufacturing company with (.

In this regard, the rate of dissociation should be measured for combinations of VH and VL in the form of Fabs. however, this means that the VH and VL domains participate equally in CD70 binding. It should be noted that he did not come. Major contributor to CD70 binding in many VH/VL combinations It is the VH domain that makes the VL domain depend on the resolution and/or Recombinant human CD70 antibodies or Fab fragments thereof described herein Binding to CD70 can also be examined by ELISA.

The CD70 antibodies described herein can also be used to detect intact cells, eg 786-0 renal carcinoma. binding to CD70 expressed on the surface of cells and other cancer cell lines can display. Binding of CD70 antibodies to CD70-expressing cells flow cytometry can be examined with In the results presented in the accompanying examples, variant 41D12 (unfucosylated It is shown that they exhibit very strong binding to CD70 on its surface. Indeed, many cell line ARGX-1 10 compared to prior art comparison antibody SGN70. exhibits strong binding.

CD70 of ARGX-101? cell lines expressing low copy number, for example others to Raji, SU-DHL-6, MHHPREBI, Mino, Mec 1, J VM-2, HH and EBC-1 cell lines. and also potently against cancer cells isolated from patients (eg CLL patients) It is particularly noteworthy that it exhibits binding, in both cases the ARGX-l 10 Its connection is significantly stronger than SGN70. Low copy number cell lines and "improved" binding to CLL patient materials for extensively recombinant CD70 It may reflect the extraordinarily high affinity of the ARGX-1107. These binding features are the ARGX-l 10 (based on 41D12) and other CD70 antibodies described herein with similar properties, not in the form of an immunoconjugate specifically bound to a cytotoxic or cytostatic moiety in the form of an IgG (for example, non-fucosylated IgG1) that exhibits potent effector function. Supporting that they have a special use in cancer treatment if they are used. is in the structure. With high affinity for recombinant CD70 and cell surface CD70° binding (i.e. higher than that achievable with prior art CD70 antibodies) with affinity) the significance of the antibodies in terms of their clinical utility in the accompanying Examples. demonstrated by experiments where cancer cell lines were added to PBMC samples. Treatment with MDX14ll and SGN70 CD70 antibodies to target targets for comparison revealed significantly more lysis in cancer cells.

The CD70 antibodies disclosed herein are directed against human CD70 and more specifically. They exhibit high affinity binding to the extracellular domain of human CD70, but Cross-reactivity with non-CD70 homologues is no exception. Actually, 27B3 and gerrnline treated variants of it here, including 4lD12 in particular An advantageous feature of many of the disclosed CD70 antibodies is their high affinity for human CD70. In addition to binding, it is also associated with monkey CD70 homologues, specifically Cheeked macaque and cynomolgus monkey cross-react with CD70 homologs.

In this regard, an inhibition of CD70-CD27 as, for example, in the accompanying example 20.2. In the ELISA, the monkey (rhesus monkey or cynomolgus monkey) compared to the human CD70 The IC50 difference for CD70 is less than 5 times, preferably less than 3 times or less than 2 times occurs, a monkey CD70 homologue of a CD70 antibody, for example the rhesus macaque and cynomolgus monkey can be considered to exhibit cross-reactivity with CD70 homologs. Human and the mine's binding affinity for CD70 and thus its blocking power is widely should be comparable.

Interaction Between CD70 and CD27 In some aspects, the CD70 antibodies or their antigen-binding antibodies provided herein fragments can bind to human CD70 with high affinity and differentiate between CD70 and CD27. they can block the interaction.

CD70 antibodies or antigen-binding fragments thereof from CD7O7 to CD27 Captured recombinant CD70 (Flag-TNC-CD70), which needs the ability to block its binding by ELISA using both directly coated CD70 and recombinant CD27-FC assessable. Interaction between CD70 antibodies, CD70 and CD27 described herein ng/ml or less, or with an IC50 of 50 ng/ml or less.

CD70 antibodies or antigen-binding fragments thereof to CD70 to CD27 its ability to block the binding of Raji cells as described in the accompanying examples. (human B-cell lymphoma) and HT1080-CD27 cells (CD27-transfected human An analysis based on co-culturing (epithelial cell line) may also be considered.

The disclosed CD70 antibodies inhibit the interaction between CD70 and CD27 in this co-culture analysis of 500 ng/ml or less or 300 ng/ml or less or 100 ng/ml or less or exhibit an IC50 of 50 ng/ml or less or 30 ng/ml or less. Preferred CD70 antibodies based on CD70/CD27 interaction in the ELISA system. showed blockage. In fact, ARGX-110 blocks the CD70/CD27 interactions with SGN70 and MDX141 1 is significantly more potent than comparison antibodies. CD70 and CD27 The interaction between tumor cell survival, proliferation and/or growth in the tumor microenvironment may be associated with immunosuppression. Therefore, it is able to bind to CD70 with high affinity. In addition, potent inhibition of the CD70/CD27 interaction is effective in some CD70-expressing cancers. or immunologic disorders, such as autoimmune co-expressing CD70 and CD27 It may contribute to the improvement of clinical outcome in the treatment of diseases and cancers.

In other embodiments, CD70 antibodies or antigen-binding fragments thereof They may bind to CD70 but not inhibit the interaction between CD70 and CD27. CD70°e CD70 antibodies that bind but do not inhibit the interaction between CD70 and CD27 are required. captured recombinant CD70 (Flag-TNC-CD70), both directly coated CD70 and can be evaluated by ELISA using recombinant CD27-FC. In the attached examples binds CD70 but inhibits CD70/CD27 interaction to a significant extent as shown Several Fabs that do not have been identified. Specifically, the Fab clone designated herein as 59D1 0 It shows strong binding to recombinant CD70 when measured by Biacore but not by ELISA. It does not block the CD70/CD27 interaction to a significant extent when evaluated. CD70*e CD70 described here that binds but does not inhibit the interaction between CD70 and CD27 antibodies still have intact antibody effector functions, i.e. from ADCC, CDC, ADCP, or ADC may have one or more of and inhibit tumor cell growth in vivo they can. CD70 antibodies that bind CD70 with high affinity but are not blocking advantageous over any other antibody format that provides a tight monovalent interaction with the cell can be used as

CD70 Epitopes10 In some aspects, the CD70 antibodies described herein are extracellular to human CD70. binds to epitopes in its domain. Kismina(kisimlar1na) is paid. Epitopes are linear, that is, have a single amino acid sequence. involving binding or conformational, that is, adjacent to various regions of the antigen involves binding to two or more amino acid sequences that are not necessarily they may be.

The CD70 antibodies described herein are distinct within the extracellular domain of the human CD70 protein. They can bind to (overlapping or non-overlapping) epitopes. For example, in the accompanying examples 1C2 and Fab71ers, designated 9E1, have clearly different, non-overlapping epitopes in human CD70. is connecting.

As noted elsewhere herein, the preferred CD70 antibody It is a particularly useful technique that no known prior art CD70 antibody exhibits. exhibits a combination of bonding properties, which are listed below: (a) human CD70°e in amino acid sequence HIQVTLAICSS (SEQ ID NO:342) attachment; (b) rhesus macaque (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus) cross-reactivity with CD70 homologues; (c) natural human CD70 and heat-denatured recombinant human CD70 don't bind.

Amino acid sequence HIQVTLAICSS (SEQ ID No:342) for any given CD70 antibody The capacity to bind human CD70*i in, for example, mouse-human described in the accompanying example 20.4 by a person of general knowledge in the art using chimeric CD70 binding analysis can be determined easily.

Cross-reactivity with monkey CD70 homologues also, eg FACS analysis, surface plasmon resonance (BiacoreTM) or using an ELISA for CD70-CD27 inhibition. easily determined by a person with general knowledge. In this regard, for example, In a CD70-CD27 inhibition ELISA as described in example 20.2, monkey (al cheeked monkey or cynomolgus monkey) ICSO difference for human CD707 versus CD707 5 of a CD70 antibody if it is less than twice, preferably less than 3 times or less than 2 times. a monkey CD70 homologue, for example rhesus macaque and cynomolgus monkey CD70 It can be assumed that it exhibits cross-reactivity with its homologs.

The CD70 antibodies described herein are both natural human CD70e and heat denatured. It may also show the capacity to bind to recombinant human CD70. In this context, the "natural" human Binding to CD70 allows a CD70-expressing cell to bind, for example, as described in the accompanying examples. Natural isolated from any of the CD70+ cell lines or even from patient material Cells expressing CD70 (for example, from CLL patients as in the accompanying examples) expressed on the surface of isolated cells), activated T cells, and the like. Indicated by binding to CD707. Consequently, binding to "natural" human CD70 is common in the art. can be easily tested by a knowledgeable person. can be tested in sequence. Significant to heat-denatured recombinant human CD70 CD70 antibody 0.6 or greater at 620 nm in this analysis to show that binding has faded. It is preferable to display a large OD.

Partial or Slow Internalization CD70 antibodies disclosed herein, including the preferred CD70 antibody 4lD12 (ARGX-110) shows partial internalization of renal carcinoma cell lines, which means 786-0 bound when antibody internalization was tested in renal carcinoma cells. even after that it stays out. In addition, CD70 antibodies have a slow rate of internalization. can also display. In this regard, if a reference CD70 antibody 9D19den (VH SEQ ID.

NO: 178; VL SEQ ID. NO: 190) a CD70 if internalized at a slower rate The antibody is considered to show slow internalization.

As illustrated in the accompanying examples, different cancer cell lines are associated with CD70 antibodies. show significant differences in terms of the degree of internalization. cancer cell lines most of them had CD70 antibody bound even after 6 hours of incubation with ARGX-1 10. less than 30% and even less than 10% internalization of it is meaningful. These results show that CD70 antibodies with strong effector function (potentiated effector function) in the treatment of CD70-expressing cancers (including variants designed for explicitly supports their usefulness. CD70lin was previously published in the scientific literature. CD70 antibody-drug immunoconjugates for the treatment of immunological diseases development is recommended. Therefore, CD70 antibodies that bind to the cell surface that its internalization is a rare event and that most CD70-expressing cell lines are conclusively that it internalizes the bound CD70 antibody to a significant extent. proving that the results presented here are extraordinarily surprising.

CD70 Antibodies from Camelidae In still other aspects, the antibodies disclosed herein or their antigen-binding fragments from a VH domain or VL domain of a species of Camelaceae. the resulting hypervariable loop or region for determining complementarity. In particular, the antibody or antigen-binding fragments of camelids, e.g. VH and/or VL obtained by effective immunization of llamas with a human CD70 antigen domains or their CDRs. hypervariable loop or complementarity determination region" overvariable loop (HV) or CDRin is a hypervariable encoded by a camelid immunoglobulin gene. an amino acid identical or substantially identical to the amino acid sequence of the loop or CDR It means you have the array. In this context, the "immunoglobulin gene" germline genes, rearranged immunoglobulin genes, as well as somatic contains mutated genes. As a result, VH or VL of a camelid species The amino acid sequence of the HV or CDR from the mature camelid conventional may be identical to the amino acid sequence of an HV or CDR found in the antibody. In this context an amino acid sequence (or small part thereof) encoded by the camelid immunoglobulin gene. Indicates a structural relationship in that it includes variants of However, this CD70 indicate a particular relationship in the context of the manufacturing process used to prepare the antibody it is not necessary.

CD70 antibodies obtained from camelids, among others, llama, arabian camel, alpaca, It can be obtained from any camel species, including vicuna, guanaco or camel.

CD70 containing camelid VH and VL domains or their CDRs antibodies are typically recombinant expressed polypeptides and chimeric polypeptides they may be. The term "chimeric polypeptide" is actually two or more peptides that do not occur adjacently. an artificial (non-naturally occurring) polypeptide created by concatenating its fragment corresponds to. Peptide encoded by two or more species, eg camelid and human Chimeric polypeptides to "species" created by concatenating their fragments are included in this definition. enters. chain CDR3) or SEQ ID: NO: or SEQ ID: one of the CDR sequences shown as NO: may contain one of the CDR sequences designated as CDR1).

In one embodiment, the entire VH domain and/or the entire VL domain is camelid It can be obtained from a species of the family. In special arrangements, camelid VH may contain the amino acid sequence shown as VH from camelids domain and/or the camelid VL domain may be subjected to protein design and wherein one or more amino acid substitutions, additions, within the camelid amino acid sequence or deleted. These engineered changes are preferably amino acids related to the camelid sequence. contains acid substitutions. These changes refer to "humanization" or "germline processing". wherein one or more in the VH or VL domain encoded by a camelid further amino acid residues to a human-encoded homologous VH or VL domain replaced by equivalent residues.

Obtained by active immunization of a camel (for example, a llama) with a human CD70 antigen. Isolated camelid VH and VL domains of the antigen-binding polypeptides of the invention It can be used as a basis for design. From intact camelid VH and VL domains one or more amino acids that diverge from the initial camelid sequence, starting substitution, insertion or deletion is possible. Some In embodiments, such substitutions, additions or deletions of the VH domain and/or VL may be present in the framework regions of the domain. In the primary amino acid sequence, this species the purpose of the changes is that the traits that are likely to be negative (for example, in a human host (so-called humanization) immunogenicity, potential product heterogeneity and/or instability domains (glycosylation, deamidation, isomerization, etc.) other positive properties (eg solubility, stability, bioavailability, etc.) could be strengthened. In other embodiments, changes in the primary amino acid sequence hypervariable Vl-l and/or VL domain may be designed in one or more of its loops (or CDR91s). These changes10 Antigen binding affinity and/or specificity likely to be enhanced or adverse Traits, such as immunogenicity (so-called humanization) in a human host, potential areas of product heterogeneity and/or instability include glycosylation, deamidation, reduction of isomerization and the like or some other positive properties of the molecule, for example, to enhance solubility, stability, bioavailability and the like.

Thus, in one embodiment, examples of the invention are, but are not limited to, SEQ ID. A VH or VL from camelids with camelid VL domains containing sequences At least one frame of both the VH domain and the VL domain compared to the or a variant CD70 antibody containing at least one amino acid substitution in the CDR region it provides.

In other embodiments, VH and VL domains from camelids (or their designed variants) and one or more constants of a non-camellar antibody domain, such as human-encoded fixed domains (or engineered variants thereof) "chimeric" antibody molecules containing In such arrangements, both VH It is preferred that both the VL domain and the VL domain be obtained from the same camelid species, for example both The VH may be from both the VL Lama glaucoma or both the VH and the VL Lama pakos (as designed prior to the addition of amino acid sequence variation). In such arrangements, VH is both VL domain active from a single animal, specifically a human CD70 antigen can be obtained from a single immunized animal.

Changes in the primary amino acid sequence of the camelid VH and/or VL domains. as an alternative to designing, extremely variable loops from individual camelid or CDRs, or combinations thereof, may be isolated from camelid VH/VL domains and a CDR to an alternative (ie non-camellar) framework, for example a human VH/VL framework can be transferred by grafting.

CD70 containing camelid VH and VL domains or their CDRs antibodies to various different types of antibodies in which both a VH domain and a VL domain are present may be in the regulations. The term "antibody" is used here in its broadest sense and Limited to these as long as they exhibit appropriate immunological specificity for the CD70 protein monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies include multispecific antibodies (eg, bispecific antibodies). Here The term "monoclonal antibody" as used is composed of substantially homogeneous antibodies. corresponds to an antibody obtained from a population, ie the individual containing the population Except for possible naturally occurring inutations where antibodies may be present in insignificant amounts is identical. Monoclonal antibodies are highly specific antibodies directed against a single antigenic site.

Also, different antibodies typically directed against different determinants (epitopes) in the antigen Unlike conventional (polyclonal) antibody preparations containing the antigen is directed at a single determinant or epitope. contains the variable domain. Examples of antibody fragments Fab, Fab', F(ab')2, bi-specific Fabs and FV fragments, di-antibodies, linear antibodies, single Chain antibody molecules from a single-chain variable fragment (SCFV) and antibody fragments. Contains multispecific antibodies generated (see contents included herein by reference) In non-limiting embodiments, VH and VL domains from camelids, or The amino acid sequence of CD70 antibodies containing their CDRs is completely or largely may contain human CH1 domains and/or CL domains. Antigen binder of the invention When the polypeptide is an antibody for human therapeutic use, the typical situation is that the antibody's wholly or substantially of the entire fixed region or at least a part thereof It has amino acid sequence. Thus, the CH1 domain, the hinge region, the CH2 domain, One or more of the CH3 domain and the CL domain (and the CH4 domain, if present) or a combination of these is completely or substantially human in terms of amino acid sequence. it could be.

Advantageously, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain, and the CL domain (and the CH4 domain, if present) completely or substantially to the human amino acid sequence is to have. of a humanized or chimeric antibody or fragment of an antibody in the context of the fixed region, the term "substantially human" is associated with a human fixed region of at least 90% or an amino acid sequence of at least 92% or at least 95% or at least 97% or at least 99% corresponds to its identity. In this context, the term "human amino acid sequence" refers to the germline, a human immunoglobulin containing edited and somatically mutated genes It corresponds to an amino acid sequence encoded by the gene. In the invention, an "all human" according to the human sequence, except in arrangements where the presence of the hinge region is absolutely essential "human" modified by the addition, deletion, or substitution of one or more amino acids Polypeptides containing sequence constant domains are also contemplated.10 The presence of a "fully human" hinge region in the CD70 antibodies of the invention to keep immunogenicity to a minimum, as well as to optimize the stability of the antibody. It might be useful.

As explained elsewhere herein, in the constant region of the heavy and/or light chain, particularly Substitution, insertion, or deletion of one or more amino acids in the Fc region. thought to be possible. Amino acid substitutions of the substituted amino acid with a different naturally occurring amino acid or a non-naturally occurring or modified amino acid may result in acid modification. Other structural modifications, such as glycosylation changes in the pattern (for example, the addition of N- or 0-linked glycosylation sites) or by deletion) is also possible. Depends on the intended use of the antibody In addition, the antibody of the invention modulates, for example, effector function in terms of its binding properties. It may be desirable to modify it to FC receptors to For example, cysteine in the Fe region residue(s) can be placed in this way, the formation of interchain disulfide bonds in this region can be provided. The homodimeric antibody formed in this way has enhanced internalization capacity. and/or increased complement-mediated cell death and antibody-induced cellular cytotoxicity. and thus may have enhanced complementary lysis and ADCC capacities. CD70 antibody can be designed. See, Stevenson et al., Anti-Cancer Drug Design 32219- 230 (1989) reference. In the invention, a cytotoxic agent, for example a chemotherapeutic agent, toxin (for example, an enzymatically active agent of bacterial, fungal, plant or animal origin) toxin or fragments thereof) or conjugated to a radioactive isotope (ie, a radioconjugate) Immunoconjugates containing an antibody as described herein are also contemplated. Pc regions can also be designed to extend the half-life as described.

In yet another embodiment, the Fc region of the antibody is responsible for antibody-dependent cellular cytotoxicity. (ADCC) mediating capacity and/or modifying one or more amino acids can be modified to increase the antibody's affinity for an Fcγ receptor by

In yet another embodiment, the glycosylation of an antibody is modified. For example, An antibody without glycosylation can be made (ie, no glycosylation in the antibody).

Glycosylation, for example, to increase the antibody's affinity for the CD70 target antigen replaceable. Such carbohydrate modifications may be, for example, one or more in the antibody sequence. This can be accomplished by changing more glycosylation sites. For example, one or more The disappearance of the variable region framework glycosylation domain thus one or more amino acid substitutions resulting in abolition of glycosylation can be done. Such deglycosylation may increase the antibody's affinity for the antigen.

Variant CD70 antibodies with an altered type of glycosylation, for example, in low amounts a hypofucosylated antibody or a nonfucosylated antibody with a fucosyl residue (Natsume et al., Drug Design Development and Therapy, Vol 3, pp. 7-16, 2009 as described in reference) or an antibody with increased bisecting GlcNac structures is also designed. ADCC antibodies of such altered glycosylation patterns typically relative to an equivalent antibody containing a "natural" human FC domain, increasing the efficacy of It has been proven to produce many times stronger ADCC. Such modifications of carbohydrates for example, a host cell with an altered glycosylation enzymatic mechanism of the antibody (Yamane-Ohnuki and Satoh, mAbs 1:3_, 230- 236, 2009). Unfucosylated with enhanced ADCC function Examples of antibodies are those produced using PotelligentTM technology from BioWa lncf.

In some embodiments, the invention includes chimeric camelid/human antibodies, particularly VH and VL. domains are entirely of the camelid sequence (for example, llama or alpaca) and the antibody may include chimeric antibodies, the remainder being entirely of the human sequence. CD70 antibodies of the VH and VL domains from camelids or their CDRs Obtained by active immunization of the VL domains with a human CD70 antigen of a camel One or more of the framework regions compared to camelid VH and VL domains may contain camelid/human chimeric antibodies, in which it contains amino acid substitution. This residues found in the VH or VL domain encoded by a human germline Sequence identity with human germline VH or VL domains replaced by equivalent residue CD70 antibodies can also be CDR grafted antibodies; where from a camelid antibody, generated by active immunization with, for example, human CD70 protein or a camel gene CDR1 (or hypervariable) derived from a camelid CD70 antibody encoded by loops) are grafted to a human VH and VL framework, the remainder of the antibody being fully human origin.

The humanized, chimeric and CDR-grafted CD70 antibodies described above, especially hypervariable obtained by active immunization of camelids with a human CD70 antigen antibodies containing loops or CDR°s, some of which are as described herein and the accompanying designed to include, and limited to, the polypeptide of interest described in the examples bacterial cells, yeast cells, mammalian cells, insect cells from prokaryotic and eukaryotic host cells, including plant cells utilizing conventional recombinant DNA manipulation and expression techniques can be easily prepared using

CD70 antibodies from camelids contain variants wherein the VH domain and/or hypervariable loop(s) or CDR(s) of the VL domain human CD707e It was obtained from a conventional camelid antibody against at least one of the hypervariable loops or CDRs (derived from camelids) one or more amino acid substitutions, additions or substitutions according to the sequence encoded by camelid It is designed to include deletion. These changes are the result of hypervariable loops/CDR31s. however, the amino acid sequence of an HV/CDRl encoded by camelgil is "largely may exhibit an amino acid sequence that is "substantially identical". Up to one or at most two amino acid sequences are incorrect with the HV/CDR encoded by match may be possible.

The camelid CD70 antibodies provided herein may be of any isotype.

Antibodies intended for therapeutic use in humans are typically IgA, IgD, IgE IgG, IgM type, often of the IgG type, if they are of the IgG type, IgG1, IgGZa and b, of one or more amino acid substitutions within the Fc moiety, or deletion or, for example, strengthening or reinforcing F c-related functionality It may be possible to make other structural modifications to reduce the

Humanization of VH and VL Domains from Camelidae Conventional camelid antibodies, the following factors described in US 12/497,239 in the preparation of antibodies that have utility as human therapeutic agents due to They provide an advantageous starting point: 1) High sequence between the Devegil VH and VL domains and their human counterparts homology %; 2) Among the CDRs of the Devegil VH and VL domains and their human counterparts10 a high degree of structural homology (i.e., human-like canonical folding structures and human-like canonical folding combinations).

Functional diversity of CD70 antibodies obtainable on camelgil (eg llama) platform also exhibits a significant advantage in this context.

In the human treatment of CD70 antibodies containing the camelgil VH and/or the camelgil VL domains. usefulness, for example, natural camelids for making them less immunogenic in a human host It can be further developed with the "humanization" of the VH and VL domains. General of humanization The goal is minimal immunogenicity of the VH and VL domains when placed in a human subject. to the antigen-binding site formed by the parental VH and VL domains. Their specificity and affinity is to produce a molecule that they protect. domain closer to the germline sequence of a human VH or VL domain involves designing changes to

Homology between a camelid VH (or VL) domain and human VH (or VL) domains determination of both camelid amino acid residues to be modified (a particular VH or in the VL domain), as well as selecting the appropriate amino acid residue(s) to replace It is an important step in the humanization process.

An approach to humanization of conventional camelid antibodies has been developed. This approach is typical of many novel camelid VH (and VL) domain sequences. somatically mutated VH (or VL) known to bind to a target antigen domains of human germline VH (or VL) sequences with human VH (and VL) consensus sequences. alignment, as well as available germline sequence information for the llama pakos.

In the following paragraphs (i) in a VH or VL domain from camelids, or selecting "camelyl" amino acid residues to substitute in a CDR thereof, and (ii) to substitute if a given camelid VH (or VL) domain is humanized Principles applicable to selecting "human" amino acid residues to substitute for is explained. This approach is SEQ ID: NO: or Camelaceae with amino acid sequences shown as of the resulting CDRs or SEQ ID: NO: or humanized one of the CDR sequences shown as VH domains from camelids with sequences shown as SEQ ID: NO: can be used for humanization.

Stage 1. Highest homology/identity with the mature camelid strain to be humanized The human (germ line) family and the member of this family are selected. A given camelid VH To identify the most closely matched human germline for the (or VL) domain A general procedure is outlined below.

Stage 2. The specific human germline family member to be used in the germline procedure is selected. Preferably, this is the highest homologous germline or a member of the same family. It is a member of another germline family.

Step 3. Amino acid for the camelid germline closest to the selected human germline Preferred locations considered for germline treatment based on use table is defined.

Stage 4. Amino deviating from the nearest human germline in the camelid germline attempts are made to replace acids; FR rather than CDR remnants of the germline process It is preferred to be done in the remains. a. Preferred are camelid and selected for use in the germline process that the amino acid in the sequence does not match the selected germline and that the same subset not found in other germlines of the class (by both V and J) encoded FR amino acids) are positions that deviate from the human germline. b. Deviation from the human germline family member selected in the germline process Locations showing the same family but used in other germlines are also discussed. receivable. c. Additional mismatches to the selected human germline (for example, additional due to somatic mutations).

The following approach is the closest match for a given camelid VH (or VL) domain. It can be used to determine the human germline: Analysis of percent sequence identity between camelid and human germline VH and VL canonical folds can be determined first, thus H1 and H2 or L1 and human germline sections with identical canonical folding combinations for L2 (and L3) identification of the family is provided. Thereafter, the relevant sequence homology grading The human germline family with the highest degree of sequence homology with the camelid variable region member can be selected. L1, L2, L3, H] and H2 hypervariable loops of the Chothia canonical classes. The identification is publicly available on the website WWW.bi0inf.0rg.uk/abs/chothia.html.page can be done with bioinformatics tools. The program's output is a lock residue in a data file shows the requirements. In these data files, key residual locations are allowed in each location. denoted by amino acids. The variable region sequence of the antibody is given as input and first The Kabat numbering scheme is determined by aligning with the consensus antibody sequence. canonical in folding analysis Martin and Thomton (Martin et al., J. Mol. Biol. 263:800-815 (1996)) a set of key residual templates obtained by an automatic method developed by used. IMGT numbering, an adaptation of the Chothia numbering scheme Using the schema, the boundaries of each frame region can be determined separately (Lefranc et al. H] and 112 or using the same canonical folding combination for L1 and L2 (and L3) family member best matched in terms of sequence homology with known specific human germline V-section can be determined. Devegil VH and VL domain framework amino acid sequences and human germline Percentage of sequence identity among corresponding sequences encoded by bioinformatics can be calculated with tools, but manual alignment of sequences can also be resorted to. Human immunoglobulin sequences, for example VBase (http://vbase.mrc-cpe.cam.ac.uk/) or Pluckthun/Honegger database (http://www.bioc.unizh.ch/antibody/Sequences/Gennlines It can be identified from various protein databases such as The camelid VH of the human sequences or A set of alignment algorithms for comparing VL domains with V regions, e.g. Available on web pages such as WWW.expasy.ch/t001s/#align are also limited Manual alignment can also be performed with a large number of sequence groups. Frame region 1, 2 of each chain and have the same canonical fold combinations as 3 and the highest degree of homology human germline light and heavy chain sequences belonging to families can be selected and related camelid variable can be compared with the region; also in FR4 with human germline JH and JK or JL regions are compared.

Identicality of residues F R1, FR2, and FR3 in the calculation of percent total sequence homology. the closest matching sequence from the human germline family, which is a combination of canonical folds. It should be known that it is examined with its use. Only residues different from the closest match or Other members of the same family with the same combination of canonical folds are scored (Note - excluding differences encoded by the primer). However, for humanization purposes, the same canonical with members of other human germline families that do not have a combination of folds Residues in identical frame regions are "negative" according to the mandatory conditions described above. may be accepted for "humanization" despite being scored. this assumption It is based on the "mix and match" approach to humanization, where Qu and FR1, FR2, FR3, and FR4 each separately with the most closely matched human germline sequence are compared and thus the humanized molecule combines a combination of different FRs. Antibodies Showing Cross Competition Monoclonal antibodies "cross-competing" with the molecules described herein, or their The antigen-binding fragments are identical to the site(s) to which the present CD70 antibodies bind. or those that bind to human CD70 in the overlapping area(s). Competing inonoclonal antibodies or antigen-binding fragments thereof, for example, by an antibody competition assay. can be determined. For example, a purified or partially purified human CD70 sample is solid. can be attached to a support. Then, an antibody compound of the present invention or its antigen binder fragment and a compound that is thought to compete with this antibody compound of the invention. monoclonal antibody or antigen-binding fragment thereof is added. one of two molecules is marked. If the marked compound and the unmarked compound are in separate and distinct fields on CD70 if it connects, it is marked whether or not the compound considered competitive is present. the compound will bind to the same level. However, if the interaction domains are identical or if it overlaps, the unlabeled compound will compete and the labeled compound that binds to the antigen amount will decrease. If the unmarked compound is present in excess, negligible the marked compound will be connected. For the purposes of the present invention, competitive monoclonal antibodies or antigen-binding fragments thereof Compounds binding to CD70 approximately 50%, approximately 60%, approximately 70%, approximately The details of the procedures for performing the competition analyzes are quite good in the art. known and, for example, by Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 567-569, ISBN 0- It can be found in reference 87969-314-2. Such assays use purified antibodies. can be done quantitatively. By titrating an antibody with itself, that is, the same for both the signal and the opponent. A standard curve is generated using the antibody. An unmarked competitive monoclonal plate of the labeled molecule of the antibody or antigen-binding fragment thereof. its capacity to inhibit its binding is titrated. The results are flagged and the desired binding The concentrations required to obtain the degree of inhibition are compared.

Preferred embodiments are F ab 2783 from llama for binding to human CD70 and including germline-processed variants thereof, especially cross-competing with antibodies and exhibiting the same combination of binding Properties These are antibodies and their binding properties are listed below: (a) binding in human CD70 by amino acid sequence HIQVTLAICSS (SEQ ID NO:342); (b) rhesus macaque (Macaca mulazta) and cynomolgus monkey (Macaca mulazta) cynomolgus) cross-reactivity with CD70 homologues; (c) binding to both native and heat-denatured recombinant human CD70.

Polynucleotides Encoding CD70 Antibodies In the invention, polynucleotide molecules encoding CD70 antibodies of the invention, as well as antigen in a host cell or cell-free expression system of the binding polypeptide and in a host cell or cell-free expression system containing the expression vector CD70 of the invention operably linked to regulatory sequences that enable its expression Expression vectors containing nucleotide sequences encoding antibodies are also provided.

Polynucleotide molecules encoding CD70 antibodies of the invention, eg recombinant DNA contains molecules. Here "nucleic acid", "polynucleotide" or a "polynucleotide molecule" The terms are used interchangeably and can be used for any single- or double-stranded to a DNA or RNA molecule and, if single-stranded, to the molecule of its complementary sequence comes the opposite. While describing nucleic acid molecules here, a certain nucleic acid A sequence or structure belonging to the molecule according to the normal rule that the sequence is provided in the 5' to 3' direction. can be explained. In some embodiments of the invention, nucleic acids or polynucleotides are "isolated". When applied to a nucleic acid molecule, the term means separated from sequences immediately adjacent in the organism's naturally occurring genome corresponds to the nucleic acid molecule. For example, "an isolated nucleic acid" is a vector, inserted into, for example, a plasmid or virus vector, or a prokaryotic or eukaryotic integrated into the genomic DNA of a cell or a non-human host organism may contain a DNA molecule. When applied to RNA, the term "isolated polynucleotide" essentially encoded by an isolated DNA molecule as described above corresponds to an RNA molecule. Alternatively, when the term is in its natural state (i.e., in cells or tissues) purified/separated from other nucleic acids with which it will be associated may correspond to an RNA molecule that has An isolated polynucleotide (either DNA or RNA) is also produced directly by biological or synthetic means and is present at the time of its production. It can represent a molecule that has been separated from other constituents.

In one embodiment, the germline treatment, herein referred to as 4lD12, is made in the invention. Nucleotide sequences encoding the VH domain and VL domain of Fabin from the llama is provided, where the VH domain is SEQ ID. To the amino acid sequence shown as No:223 and VH domain SEQ ID. It has the amino acid sequence shown as NO:241. Choice SEQ ID in one embodiment. VH with amino acid sequence shown as NO:223 The nucleotide sequence encoding the domain SEQ ID. Contains the nucleotide sequence shown as No:344 and SEQ ID. encoding the VL domain with the amino acid sequence shown as NO:24] nucleotide sequence SEQ ID. It contains the nucleotide sequence shown as NO:345.

The VH of the F abi obtained from the germline treated llama expressed here as 57B6 The nucleotide sequences encoding the VL domain and the VL domain are also described, wherein VH domain SEQ ID. It has the amino acid sequence shown as NO:274 and the VH domain SEQUENCE ID. It has the amino acid sequence shown as No:276. SERIAL in a preferred embodiment ID. The nucleotide sequence encoding the VH domain with the amino acid sequence shown as NO:274 SEQ ID. Contains the nucleotide sequence shown as No:346 and SEQ ID. as No:276 SEQ ID of the nucleotide sequence encoding the VL domain with the amino acid sequence shown. No:347 contains the nucleotide sequence shown as

The VH of Fabin obtained from the germline treated llama expressed here as 59Dl 0 The nucleotide sequences encoding the VL domain and the VL domain are also described, wherein VH domain SEQ ID. It has the amino acid sequence shown as No:275 and the VH domain SEQUENCE ID. It has the amino acid sequence shown as NO:277. SERIAL in a preferred embodiment ID. The nucleotide sequence encoding the VH domain with the amino acid sequence shown as NO:275 SEQ ID. Contains the nucleotide sequence shown as No:348 and SEQ ID. as NO:277 SEQ ID of the nucleotide sequence encoding the VL domain with the amino acid sequence shown. No:349 contains the nucleotide sequence shown as

For the recombinant production of a CD70 antibody according to the invention, a recombinant polynucleotide can be prepared (using standard molecular biology techniques) and a selected a replicable for expression in a host cell or a cell-free expression system is inserted into the vector. Suitable host cells are prokaryotic, yeast or higher eukaryotic cells. particularly mammalian cells. Useful mammalian host cell Examples of lines are monkey kidney CV1 line transformed with SV40 (CCS-7, ATCC CRL 1651); human embryonic kidney line (bottom for growth in suspension culture) baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-; canine kidney cells (MDCK, ATCC CCL 34); bovine/rat liver cells (BRL 3A, ATCC CRL ; human liver cells (Hep G2, HB ; TR1 cells cells; and human hepatoma line (Hep 02), as well as DSM's PERC-6 cell line. This Expression vectors suitable for use in each of the host cells are also generally available in the art. known. should be specified. An expression encoding an antigen-binding polypeptide of the invention into expressly excluded from the definition of the entire human "host cell" into which the vector has been inserted. are held.

Antibody Production In an important aspect, there is no information about the production of a CD70 antibody of the invention. A method is also provided, said method comprising the polynucleotide encoding the CD70 antibody. (for example, an expression vector) to a host cell (or cell-free expression system) culture under conditions that enable expression of the CD70 antibody and involves recovery of the expressed CD70 antibody. This recombinant expression including monoclonal antibodies intended for therapeutic use in humans It can be used in the large-scale production of suitable CD70 antibodies. In vivo therapeutic use Suitable vectors for the large-scale manufacture of recombinant antibodies suitable for cell lines and manufacturing processes are generally available in the art and are known to those skilled in the art.

As noted elsewhere, the CD70 antibodies provided herein in particular, in particular Fab Antibodies based on these exhibit properties that are particularly useful for commercial manufacturing on a large scale. Specifically, it was obtained in a commercial scale recombinant expression system. The extraordinarily high production yield (>4g/L) that can be produced can dramatically reduce production costs. will reduce.

Preferred embodiments test at 37°C as well as various freeze-thaw cycles once again outstanding for the commercial manufacture and storage of a clinical product. Various variants of human germline processed including 27B3 with itself It exhibits improved thermal stability in comparison.

Here, an isolated antibody that binds to human CD70 or its antigen-binding fragment is also disclosed, wherein said antibody or fragment is H6, HI 8, H24, H31, H56, where at least one amino acid at a selected Kabat position is substituted with another amino acid provided it is SEQ ID. A heavy one corresponding to the amino acid sequence specified in NO: 1783 contains the chain variable domain.

This antibody, or antigen-binding fragment thereof, is SEQ ID. amino acid specified in NO:178 An antibody or antigen containing a heavy chain variable domain having the sequence may exhibit higher thermal stability than the binder fragment.

at least one amino acid at a Kabat position selected from the group consisting of L879 SEQ ID, provided it is substituted with amino acid. NO:20] amino acid a CD70 antibody containing a light chain variable domain corresponding to the sequence The antigen binding fragment is also described.

This antibody or antigen-binding fragment thereof is SEQ ID. Amino acid specified in No:2013 An antibody or antigen containing a heavy chain variable domain having the sequence may exhibit higher thermal stability than the binder fragment.

The CD70 antibody or antigen-binding fragment thereof may include: One or more of the Kabat locations selected from the group consisting of H] 10 and H1 12 SEQ ID containing amino acid substitution. to the amino acid sequence specified in NO:178 having a heavy chain variable domain; and and one or more amino acids at Kabat positions selected from the group consisting of L87 SEQ ID containing the substitution. A light weight with the amino acid sequence specified in No:201 chain variable domain.

This antibody, or antigen-binding fragment thereof, is SEQ ID. amino acid specified in NO:178° A heavy chain variable domain with the sequence and SEQ ID. amino acid specified in NO:201° An antibody or antigen containing a light chain variable domain having the sequence It may exhibit greater thermal stability than the binder fragment.

Therapeutic Usefulness of CD70 Antibodies CD70 antibodies or antigen-binding fragments thereof provided herein They can be used to inhibit the in vivo growth of cells and thus CD70 They are useful in the treatment of cancers that express

Thus, other aspects of the invention inhibit tumor cell growth in a human patient. relates to the methods of treating or preventing cancer, a CD70 antibody or antigen as described herein to a patient in need of it. involves administering a therapeutically effective amount of the binding fragment.

In another aspect of the invention, CD70-expressing tumor cells in a human patient a CD70 antibody as described herein for use in inhibiting the growth of or the antigen-binding fragment is provided.

In yet another aspect of the invention, it is used to treat or treat cancer in a human patient. a CD70 antibody or antigen as described herein for use in the prevention of linker fragment is provided.

Use the CD70 antibodies described herein to be used to inhibit the growth of the preferred cancers renal cancer (eg, renal cell carcinoma), breast cancer, brain tumors, chronic or acute leukemias, eg acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas (eg, Hodgkin and non-Hodgkin lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma), nasopharynx carcinomas, melanoma (eg, metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tubes carcinoma, endometrium carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, esophageal cancer, thin bowel cancer, endocrine system cancer, thyroid gland cancer, parathyroid gland cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood cancer tumors, bladder cancer, kidney or urethra cancer, renal pelvis carcinoma, central nerve system (CNS) neoplasm, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary gland adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer and includes mesothelioma.

The CD70 antibodies described herein are associated with the presence of CD70-expressing tumor cells. a tumor-forming disorder that manifests itself, for example, renal cell carcinomas (RCC), eg clear cell RCC, glioblastoma, breast cancer, brain tumors, nasopharyngeal carcinomas, non-Hodgkin lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt lymphoma, anaplastic large cell lymphomas (ALCL), multiple myeloma, cutaneous T-cell lymphomas, nodular small discrete cell lymphomas, lymphocytic lymphomas, peripheral T-cell lymphomas, Lennert lymphomas, immunoblastic lymphomas, T cell leukemia/lymphomas (ATLL), adult T cell leukemia (T-ALL), entroblastic/centrocytic (cb/cc) follicular lymphoma cancers, diffuse large cell B-derived lymphomas, angioimmunoblastic lymphadenopathy (AILD)-like T-cell lymphoma, HIV associated abdominal cavity lymphomas, embryonal carcinomas, rhino-pharynx undifferentiated carcinomas (eg, Schmincke tumor), Castleman disease, Kaposi sarcoma, multiple myeloma, Waldenstrom macroglobulinemia, Mantle cell lymphoma, and It may also be used to treat a subject with other B-cell lymphomas. Special arrangements are made for any of the above listed cancers, including F ab derived from llamas. including germ line procedure It relates to treatment with an antibody containing one of its variants. To specifically point out, any of the above listed cancers fused with constant regions of human IgG1 with an antibody containing Fab regions of 41D12 (germline-operated variant of 27B3) curable. In the latter embodiment, the human IgG1 constant region antibody effector It can also be designed separately to maximize its functions (for example, point mutations by adding) or 41D12-IgG1 antibody again to strengthen the effector function of the antibody. may not be fucosylated.

As noted elsewhere, the CD70 antibodies disclosed herein, in particular 27B3 and its10 Gerrn-line-treated variants express CD70 in a low copy number on the cell surface. human cancer cell lines, such as the large B-cell lymphoma cell line SU-DHL-6, chronic lymphocytic leukemia cell line JVM-Z, cutaneous T-cell lymphoma cell line HH, gastric carcinoma cell line MKN-45, lung carcinoma cell lines A549 and EBC-1, melanoma cell lines WM852 and WM793, glioblastoma cell line GaMG and ovarian carcinoma cell lines lines OAW-42, SKOV3 and OVCAR37 show particularly strong binding. really, this Its affinity for low copy number cancer cell lines is SGN70 and Significantly stronger than prior art comparison antibodies such as MDX1411 is high.

"Improved" affinity for low copy number cancer cell lines directly with the clinical treatment of the corresponding cancers, as demonstrated by challenge experiments. is related. A low copy number cell line (for example, diffuse large B-cell lymphoma) cell line SU-DHL-ö) to newly isolated PBMCs from healthy donors When loaded, the cancer cell line cells are detected by the exemplary CD70 antibody ARGX-1 10. consumed on demand.

Thus, another aspect of the invention is the need for it to treat or prevent cancer. a CD70 antibody or antigen-binding agent as described herein. Relating to a method comprising administering a therapeutically effective inictarine of the fragment, wherein the cancer is a cancer that expresses CD70 in a low copy number.

The invention is also described for use in the treatment or prevention of cancer in a human patient. A CD70 antibody or antigen-binding fragment as described herein is provided, wherein the cancer is a cancer that expresses CD70 in a low copy number.

In each of these aspects, low copy number cancer preferably large B-cell lymphoma, chronic lymphocytic leukemia, cutaneous T-cell lymphoma, gastric cancer, lung cancer, melanoma, selected from the group consisting of glioblastoma and ovarian cancer. Special embodiments include Fab 27B3 from llama of low copy number cancers or one of its germline-treated variants, specifically 4lD12° (ARGX-110) to be treated with an antibody containing Specifically, the above any of the listed low copy number cancers with constant regions of human IgG1 Fab regions of fused 41D12 (germline-operated variant of 27B3) can be treated with an antibody containing

The CD70 antibodies described herein are immunologic10 manifested by CD70 expression. It is also useful in the treatment of disorders. Certain of these immunological disorders Examples include: rheumatoid arthritis, autoimmune deinyelinating diseases (eg, inultiple sclerosis, allergic encephalomyelitis), endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosis, myasthenia gravis, Grave's disease, glomerulonephritis, autoimmune hepatologic disorder, inflammatory bowel disease (eg, Crohn's disease, ulcerative colitis, Celiac disease), anaphylaxis, allergic reaction, Sjogren's syndrome, type I diabetes mellitus (diabetes), primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schinidt syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressier syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemtigus vulgaris, pemtigus, dermatitis herpetiformis, ringworm, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal motility disorder, sclerodactyly and telangiectasia), male and female autoimmune infertility, ankylosing spondylitis, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture syndrome, Chagas disease, sarcoidosis, rheumatic fever, asthma, recurrent miscarriage, phospholipid, farmer lung, erythema multiforme, post-cardiotonin syndrome, Gushing's syndrome, autoimmune chronic active hepatitis, bird feeder lung, toxic epidermal necrolysis, Alport syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transition reaction, Takayasu arteritis, rheumatic polymyalgia, temporal arteritis, cystozoiniasis, giant cell arteritis, acariasis, aspergillosis, Sampter syndrome, eczema, lymphomatoid granulomatosis, Behçet's disease, Caplan syndrome, Kawasaki disease, dengue, encephalomyelitis, endocarditis, endomyocardial tibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic fasciitis, Shulman syndrome, Felty syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch cyclitis, IgA nephropathy, Henoch-Schonlein purpurasi, graft versus host disease, transplant rejection, cardiomyopathy, Eaton-Lambert syndrome, recurrent polychondritis, cryoglobulinemia, Evan syndrome and autoimmune gonadal insufficiency, B lymphocyte disorders (for example, systemic lupus erythematosus, Goodpasture syndrome, rheumatoid arthritis and type I diabetes), Thl - lymphocytes (eg, rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis or graft versus host disease) or Th2- lymphocytes (for example, atopic derrnatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn syndrome, systemic sclerosis or chronic graft versus host disease), Churg Strauss syndrome, microscopic polyangiitis and Takayasu arteritis. Special arrangements are made for any of the immunological disorders listed above. germline processing including the obtained F ab It relates to treatment with an antibody containing one of its variants. specifically point out if necessary, any of the cancers listed above are associated with constant regions of human IgG1. Fab regions of fused 4lD123 (germline-operated variant of 27B3) can be treated with an antibody containing In the last mentioned embodiment, the human IgG1 constant region The antibody can be further designed to maximize effector functions (e.g. point by adding mutations) or the 41D12-IgG1 antibody again interferes with the antibody's effector function. It may not be fucosylated to strengthen it.

As used herein, the term "treating" or "treatment" is a symptom, disorder, slowing, interrupting, stopping, controlling illness or disease means to be taken, cured, terminated, lessened in severity, but the total absence of all symptoms, ailments, or disorders associated with the disease It is not necessary to include it.

As far as therapeutic use in humans is concerned, the CD70 antibodies described herein are therapeutic. A needing human subject may also be given an "effective amount". The term "effective amount" is a CD70 antibody in the treatment of disease when given to a human patient in a single or multiple dose. corresponds to the amount or dose that provides therapeutic efficacy. CD70 antibody therapeutic Effective amounts range from about 0.1 mg/kg to about 20 mg/kg per single dose. may contain some A therapeutically effective amount of CD70 for each individual patient A biomarker of the antibody, for example, cell surface CD70 in tumor tissues, or a symptom, eg tumor regression, etc. health professionals by monitoring the impact on can determine. The amount of antibody administered at a given time point, either alone or as a Whether used in combination with another therapeutic agent, CD70 throughout the course of treatment It can be varied to allow optimal amounts of antibody to be administered.

The CD70 antibodies disclosed herein, or pharmaceutical compositions containing these antibodies, as a combination therapy in combination with any other cancer treatment grant is also considered.

Antibodies that internalize too little to deplete CD70-expressing cells to use A surprising finding described here is CD70ie expressed on the surface of cancer cell lines. very little internalization of bound CD70 antibodies. In the previous art This is surprising, as CD70 has been described as an "internalizing target". is the result. Although CD70 antibodies that bind between different types of cancer cells are completely Although there is variation in the degree of internalization, no type of cancer cell It has not been observed to "fully internalize" the CD70 antibody. For example, previously internalizing renal cancer cell lines actually bound antibody ARGX-110° It has been shown that they internalize about 70% of the maximum. Many other cancer cells internalizes the azine.

The variation in the degree of internalization among different cancer cell lines is great. The extent depends on the disease indication and is similar when different CD70 antibodies are used. results are not observed. However, when antibody ARGX-11 is used, some cell It has been specially observed that there is very little internalization in the lines. many cancers low degree of internalization observed in the cell, in vivo depletion of cancer cells having potent effector function (for example, killing them or inhibiting their growth) Provides justification for the use of CD70 antibodies. Many cancers are caused by binding CD70 antibodies. Because they are relatively poorly internalized in the cell, the antibody will remain attached to the cell surface. and thereby effector functions through interactions with the immune system of a human patient. (ADCC, CDC, ADCP).

Accordingly, herein, CD70-expressing cells are depleted in a human patient. A method is described for treating said patient with a human CD703 binding comprising administering an effective amount of the antibody, wherein said CD70-expressing cells are 30% or less, 25% or less of the antibody in question after a 6 hour period. exhibits internalization, wherein said antibody is composed of ADCC, CDC, and ADCP. exhibits at least one effector function selected from the group.

In this method, cells expressing CD70 antibody (with active effector functions) used for consumption. Such depletion of CD70-expressing cells is therapeutic or prophylactic therapy, for example, treatment or prevention of CD70-expressing cancers or Essential for the treatment or prevention of CD70-associated immunological disorders can create. Precise mechanism for depletion of CD70-expressing antibodies CD70 This may depend on the type of effector functions the antibody exhibits, but the weak CD70e on the cell surface of the CD70 antibody with its internalization affinity) will also depend on the binding.

In some embodiments, cells expressing CD70 are cancer cells expressing CD70. Specifically, CD70-expressing cancer cells Burkitt lymphoma B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, pancreatic carcinoma, gastric carcinoma, glioblastoma and lung It may be a type of cancer selected from the group consisting of carcinoma. From each of these cancers The cell lines obtained contained 30% of the bound CD70 antibody in a 6 hour period. or less has been proven to internalize. In another embodiment, CD70-expressing cancer cells Burkitt lymphoma, large B-cell lymphoma, Hodgkin lymphoma, Hodgkin female lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, gastric carcinoma, glioblastoma and a type of cancer selected from the group consisting of lung carcinoma. Each of these cancers CD70 antibody bound within a 6 hour period of cell lines obtained from one cancer cells expressing Burkitt lymphoma, large B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, gastric carcinoma and lung carcinoma It may be a selected type of cancer. 6 of the cell lines derived from each of these cancers Intemalize 10% or less of the bound CD70 antibody in a one-hour period. it has been proven..

In other embodiments, cells expressing CD70 are associated with bound CD70 antibodies. exhibiting no detectable internalization (notably a 6-hour ARGX-110 internalization was not observed in the time period) CD70-expressing activated T may have cells. by CD70+ activated T cells of bound CD70 antibodies This observation that they are not internalized indicates strong effector function (ADCC, CDC, or CD70 antibodies with ADCP), for example, but not limited to, ARGX-11 eg rheumatoid arthritis, psoriasis, SLE, etc. CD70+, which includes autoimmune disorders such as its use in the treatment of immunological diseases mediated by activated T cells. strongly supports.

In the embodiments listed above, essentially any CD70 antibody can be used. In particular embodiments, antibody Raji, SU-DHL-o, MHHPREB1, Mino, Mecl, JVM-Z, MKN-45, 30% or more after a 6 hour period in a selected CD70-expressing cancer cell line Any CD70 that has been experimentally shown to exhibit or less Could be antibodies. In fact, the cell line internalization analysis described in the accompanying examples, with CD70 antibodies with potent effector function or with CD70 antibody-drug conjugates whether certain disease indications (explained below) can be targeted. CD70 is suitable for identification and also for the treatment of certain disease indications. It can be used as a screening for the identification of antibodies.

Thus, herein, CD70-expressing cancer cells are depleted in a human patient. an effective amount of an antibody that binds to human CD70 in the subject A method of administering is also disclosed, wherein said antibody is Raji, SU-DHL-6, Granta 519, a CD70-expressing cancer selected from the group consisting of Rec-1 and EBC-1 exhibits 30% or less internalization in the cell line after a period of 6 hours, and wherein said antibody is at least one selected from the group consisting of ADCC, CDC and ADCP. exhibits an effector function. In one embodiment, CD70-expressing cancer in a human patient The antibody to be used in depletion of cells is Raji, SU-DHL-ö, Granta 519, Rec-1, Mecl, A CD70-expressing cancer selected from the group consisting of JVM-2, MKN-45, A549 and EBC-1 exhibiting 10% or less internalization at the cell line after a 6-hour period, and also exhibiting at least one effector function selected from the group consisting of ADCC, CDC, and ADCP. Could be antibodies. It binds with exceptionally high affinity to human CD70 in specific embodiments. Any of the CD70 antibodies may be used. Obtained from laina in preferred embodiments F ab 27B3, or one of its germline-treated variants, especially containing an antibody can be used. Especially in preferred embodiments 41D12 fused with constant regions of human IgG1 (gerrnline processed of 27B33) variant) An antibody containing Fab regions is used. In the last-mentioned embodiment, human (for example, by adding point mutations) or the 41Dl2-IgG1 antibody is still the effector of the antibody. It may not be chocosylated to strengthen its function. CD70 antibodies that are minimally internalized in depletion of CD70-expressing cells. In this method, the logic behind its use is based on the effector functions of the antibody. of the antibodies used, the CD70 antibody to a pharmaceutical substance, cytotoxic substance,10 immunoconjugates to which it has been bound to cytostatic agent, drug moiety, etc. It is preferable not to. In fact, a particular advantage of this method is the antibody-drug conjugates are not needed, which is both in terms of patient safety. (avoiding the administration of potentially toxic substances), both economically (antibody-drug It is desirable to avoid costly synthesis of conjugates.

Antibody in Killing Cells with Significant CD70 Antibody Internalization Use of Drug Conjugates In the present examples, the degree of intemalization of CD70 antibodies from cell to cell There are significant differences and these differences are largely due to the nature of the CD70 antibody. cell type rather than cell type (although when ARGX-1 10 antibody is used) although the differences are particularly pronounced). Significant for bound CD70 antibody a degree of intemalization, ie 30% or more, preferably 40% or more, after 6 hours more, more preferably 50% or more, or even 60% or more internalization An alternative therapeutic strategy is the CD70 antibody- It can be based on the use of drug conjugates.

Thus, we are talking here about depletion of CD70-expressing cells in a human patient. an antibody that binds to human CD70 and a cytotoxic or cytostatic drug fragment to the subject patient a method comprising administering an effective amount of an antibody-drug conjugate containing is described, wherein said cells expressing CD70 are recovered after a period of 6 hours. 30% or more, 40% or more for said antibody binding to human CD70, In particular embodiments, cells expressing CD70 antibody-drug over a 6 hour period. In this method, the CD70 antibody-drug conjugate is used to deplete CD70-expressing cells. used. Such depletion of CD70-expressing cells is therapeutic or prophylactic. treatment, for example, treatment or prevention of CD70-expressing cancers or with CD70 It can form the basis for the treatment or prevention of associated immunological disorders. CD70 The precise mechanism for depletion of expressing antibodies is that the antibody-drug conjugate may depend on its nature, i.e. whether the "drug" part is cytotoxic or cytostatic, but on the cell surface of the CD70 antibody, with strong internalization of the bound antibody. It will also depend on its strong (high affinity) binding to CD70.

In some embodiments, cells expressing CD70 are cancer cells expressing CD70. Specifically, cancer cells expressing CD70 include cutaneous T-cell lymphoma, consisting of multiple myeloma, renal cell carcinoma, astrocytoma, melanoma, and ovarian carcinoma may be from a cancer type selected from the group. Cell from each of these cancers lines of more than 30% of the bound CD70 antibody in a 6-hour period. It has been proven to internalize. In another embodiment, cancer cells expressing CD70 selected from the group consisting of renal cell carcinoma, astrocytoma, melanoma and ovarian carcinoma. It could be a type of cancer. 6-hour cell lines from each of these cancers over a period of time, more than 507% of the bound CD70 antibody was internalized. has been proven.

In particular embodiments of this aspect of the invention, the antibody-drug conjugate human CD70,e to any of the CD70 antibodies described herein that bind with exceptionally high affinity can withstand. Fab from antibody-drug conjugation in preferred embodiments 27B3 or one of its germline-treated variants, specifically 41D12 containing an antibody. Antibody-drug intended for therapeutic use in humans properties of their conjugates, particularly the nature of the "drug" moiety, eg cytotoxic or cytostatic pertaining to being a substance and conjugation of the drug fragment to the CD70 antibody fragment properties are generally known in the art. Examples of CD70 antibody-drug conjugates, Pharmaceutical Compositions The scope of the invention includes CD70 antibodies of the invention or their antigen binding containing one or more of its fragments or a combination thereof. pharmaceutically formulated with a pharmaceutically acceptable carrier or excipient contains compositions. These compositions contain one or a combination of CD70 antibodies. (for example, two or more different).

Techniques for formulating antibodies for therapeutic use in humans are well within the art. known and, for example, Wang et al., Journal of Phannaceutical Sciences, Vol. 96, p. one- 26, 2007 . Examples The invention is better understood by reference to the following non-limiting experimental examples. will be understood. Example 1: Immunization of Llamas Immunization of llamas and collection of peripheral blood lymphocytes (PBL), as well as followed by RNA extraction and amplification of antibody fragments by De Haard et al. was carried out in sequence. Two llamas expressing CD70 and two slides were immunized with CD70-expressing Raji cells (ATCC-CCL-86). cells each newly prepared for immunization and CD70 expression was confirmed by FACS analysis. Llamas approximately 107 injected intramuscularly into the neck weekly for six weeks. immunized with living cells. Neck a few centimeters from the cellular immunization area Incomplete Freund's Adjuvant was injected into his muscles.

10 ml blood samples before and after immunization to investigate immune response gathered. Llama serums before immunization (day 0) and after (day 55 or 69) Test for the presence of recombinant CD70 antibodies (Flag-TNC-CD70) with ELISA was done. Distinction between conventional and heavy chain antibodies as detection antibodies goat anti- It should be noted that llama IgGl/Z (Bethyl, A160-100P) is used. Results in Figure 1 is shown.

Three to four days after the last immunization, a Ficoll-Paque was used to isolate PBLs. To prepare the gradient and RNA°y1, Chomczynski P et al., Anal. biochem. 162:156- ml of blood was collected. On average, a yield of 450 µg of RNA was obtained, of which 80 µg. a representative amount of random cDNA synthesis followed by VHCH] , VÄCÄ and VKCK gene sections was used for amplification. Example 2: Creating a Library Independent VKCK and VKCK libraries after 25 cycles with unlabeled primers using a two-step PCR with 10 cycles of these primers with the marked variant (De Haard H et al., Biol. Chem. 274, 1999). VHCHl libraries are the same was done in parallel using the approach. The dimensions of each library separately are 107 and 10[0 happened between cfu. After that, the light chains of the VKCK and VKCK libraries are separated separately. recloned into the vector expressing VHCH1 as "Lambda" and "Kappa", respectively. The lama Fab-library was created. Alternatively, digested VHCH1 amplicons Directly to VKCK and VÄCÄ libraries, avoiding creating a VHCHl library was cloned. Light chain shuffling is generally better than heavy chain shuffling. to generate pre-cloned light chain libraries as they provide good affinity variants. and heavy chain amplicons directly into these primer repertoires.

The final phage display libraries were between 5X108 and 2)(109 cfu. Using PCR quality was routinely assessed by analyzing the percentage of full-length F ab containing clones in libraries. control was carried out. Example 3: Selections and Scanning On captured recombinant CD70 (Flag-TNC-CD70) or directly coated CD70 Two to three rounds of phage selection were performed using standard protocols. Bound phage elution made with trypsin.

Randomly separate selected phage transfected into TG1 to grow in 96-well plates. colonies were selected. Fab-containing after IPTG induction according to standard protocols periplasmic fractions (peris) were prepared. For all elections and all four lamas Inhibition of the interaction between CD27 and CD70 as determined by inhibition ELISA* Various clones have been found to be capable of expressing FAs capable of expression (Fig. 2 and 3). In this analysis, Flag-TNC-CD70 was captured with an anti-Flag mAb and binding to CD27-Fe. In the absence of CD70 Fabs (Figures 2 and 3) or CD70 mAbs (Figure 4), or Biotinleninis was detected through an anti-CD27 mAb and strep-HRP in the presence of strep.

with a VH° identical to 9D] in Figure 2B, but with a slightly different light chain 27B3 appears to more effectively block CD27° binding to CD70° It can be seen that when measured with Biacore (data not shown) this Fab had better It is parallel to the fact that it has a separation rate. CD70 inhibition in Table 3 ELISA,sinda blocking potency (expressed as 1C50) for the various llama-human chimeric mAbs tested When regulated as llama-human IgGl it improves further, yielding an IC50 of 0.25 nM, other mild chain mAb 9D] is 3 times less potent (Fig. 3 of the llama CD70 Fables). blocking activity (in the inhibition ELISA, see white bars) and CD70 binding (linking ELISA, black bars). Binding In ELISA, Flag-TNC-CD70 is captured with an anti-F lag mAb and the binding of Fab7s is Fd. An anti-myc-HRP mAb recognizing the MYC tag fused to the C-terminus of the fragment detected using Known monoclonal antibodies to CD70 for comparison The MDX69A7) described in Figure 5A and SB in the document was tested in parallel. Pure bonding at ELISA Several Fab clones have been identified from the non-blocking laina showing interestingly, it is a chimeric 1gG9, which does not have blocking activity as F ab is the 59D10 clone that is antagonistic when transformed (see Table 3). from llama Here are the amino acid sequences of the VH and VL domains of the binding and blocking Fab clones. shown elsewhere (Tables 6 - 9).

Table 3: Comparison of individual SIMPLE antibodies (mAbiler) and SGN70 and CD70 Inhibition Using Recombinant Human CD70 For MDX69A7 Blocking Power Measured in ELISA and Expressed as [C50 (ng/ml) mAb IC50 (ng/ml) 9D] 109 5F4 296 9E1 1400 902 40 9B2 126 4D2 254 24D4 34 24B6 420 27B3 38 19G10 75 57B6 154 59D10 107 Rhesus monkey recombinant CD70 (FLAG-TNC-rhesus monkey) in mAbs 27B3 and 57B6 tested for their capacity to inhibit the interaction between monkey CD70) and CD27. was done. The IC50 value calculated by the inhibition ELISA described above is 150 and It was 135 ng/ml. Example 4: Affinity for Human and Rhesus Monkey CD70 Dissociation rates of purified anti-CD70 blocking Fabs from llama Biacore was calculated using Recombinant human CD70 was fixed on a CMS Biacore chip.

Fixation according to a method provided by Biacore and using the NHS/EDC kit (Biacore AB) carried out using: 10 mM acetate buffer pH 1.5 after chip activation A 50 tig/ml solution of recoinbinant CD70 was prepared in luli of this solution (50 ng). was injected, revealing a surface density of approximately 1000 RU.

Fabs for CD70 mAbs ARGX-1 10, MDX2H5 and SGN70 by trypsin digestion (SGN70 and MDX69A7 are described elsewhere here, also as MDX1411 Fab Hepes-buffered saline (0.1M) at a concentration of approximately 100-400 tig/ml Hepes diluted 6 times in it.

They were injected (30 µl) and passed through flow cells at a flow rate of 30 µl/min. Fabsnin After binding to CD70, the dissociation rate was monitored for 10 minutes. after separation Flowing cell surfaces were regenerated by injecting 5 ;41 10 mM NaOH. Sometimes to the affinity of Fabi Therefore, a large number of NaOH injections are needed to renew the surfaces.

The separation rate analysis was performed by applying the BlAevaluation software. Empty flows first The sensogram was subtracted from those obtained with the coated flow cell. Later Fit Kinetics The separation rate was determined in a time interval of 10 minutes using the application and Kd value was calculated. In addition, the dissociation rate of CD27 for CD70 was determined. Separation rates It is summarized in Table 4.

All Fabs tested from llamas ranged from 0.4-4.8 X 10'4 s1 to Biacore cleavage. human recombinant CD70°e and rhesus monkey recombinant CD70°e when looking at their rates it obviously binds with very low dissociation rates (ie, high affinity). llama Dissociation rates of the resulting Fabls for human CD70 Fab from the reference mAb (7-30 X 10'4 s") than for Comparable dissociation with that of CD27 in terms of interaction of the Fab51s with CD70. It has been shown that they have speeds (0.8 X 10*4 delete). Decomposition using Biacore It should be noted that the lower limit for the measurement of velocities is around 0.4 X 10'4 s'1. Therefore The results obtained from llamas with dissociation rates close to this limit when evaluated with Biacore. Fabs may indeed exhibit an even higher affinity for CD70.

Table 4: Human or Rhesus Fabs from Llamas and Reference Fabs Decomposition Rates for Monkey CD70 Fab km (s`i) tested x E`4 human CD70 km (5”) x E`4 rhesus monkey CD70 SGN70-Fab 7.0 MDXZHS-F ab 17 No binding MDX69A7-Fab 30 Tested Fab koff (s'l) X E'4 human CD70 koff (5") x E'4 rhesus monkey CD70 CD27 0.8 1C2 0.4 3.2 9D] 4.8 2.0 902 1.8 1.8 5F4 0.8 5.0 9151 1.9 2.0 9B2 2.8 6.1 4D2 3.3 2.9 27B3 0.8 24133 0.8 33D8 4.5 24F2 1.6 24B6 1.0 19010 0.8 8812 3.6 45Bl2 2.1 45D9 2.1 45F8 2.0 57B6 1.0 0.4 59D10 13.9 No binding Example 5: Binding of Chimeric Lama-Human CD70 mAbs to CD70 Positive Cells VH and VL human IgG1 constant regions of Fab clones obtained from said llama and human Cklya (all variable light chain regions are of lambda origin) fused and WO produced as antibodies. Chimeric llama-human mAbs were purified in Protein A, then gel filtration was done. 786-0 cells with chimeric llama-human CD70 inhibitory mAbs in 1/5 dilution series (20 ng/ml - 0.25 ng/ml) was incubated and binding of mAb°s to cells was an anti-human Fc- Using FITC antibody (conjugate AF006 diluted 1/500 from Binding Site) detected. 10,000 cells/condition Fluorescence was measured using a flow cytometer and averaged fluorescence is marked. The results are shown in Figure 5. Most mAb71s are highly affinity Connected to cells 786-0. Just like Fab, it has the best dissociation rate and blocking power SIMPLE antibody demonstrated to be unable to recognize CD70* expressed on the 1C2 cell surface (data not shown) and therefore not studied further. SIMPLE antibody 2783 0.24 nM with an EC50, it has the best affinity for the cell-bound receptor.

In another experiment, MHH-PREB-1 cells (105 cells) from NHL1 were chimeric lama- from human CD70 inhibitory mAbs with a concentration gradient (20 ng/ml to 0.25 ng/ml) incubated and binding of mAbs to cells using anti-human IgG-FITC antibody Example 6: Chimeric Lama-Human CD70 mAb in Co-Culture Experiments inhibition For determination of blocking power in a cell-based assay, Wyzgol et al. transfected with fibrosarcoma cell line HT1080 trimeric recombinant CD70 It releases 1L-8 when bound, and blocking this interaction with a neutralizing mAb is low. can be measured by cytokine levels. The assay allows the molecule to be expressed on the cell surface and Therefore, although it is more suitable than soluble CD70, B-cell lymphoma Raj i cell A modified version of the line acting as a CD70 source is used.

Raji cells were cultured and CD70 expression was checked by FACS analysis. Later on cells in a 96-well tissue culture plate (50,000 cells/well) anti-CD70 Mixed with mAb71s and incubated for 1 hour at room temperature. HT1080-CD27 cells added (10,000 cells/well) and mixed well. Co-cultures were transferred to the incubator and grown overnight at 37°C. Supernatants were collected and their IL-8 contents analyzed by ELISA. was done. Results for SIMPLE antibodies are shown in Figure 6 and reference CD70 are compared with mAbs. lC50 values for chimeric llama-human CD70 mAbs, (See Table 5). SIMPLE antibodies and 902 (20 pM 3 times stronger than the comparative MDX69A7 (1 nM). Hence the chimeric lama-human Exceptionally potent in blocking the interaction of mAbs between CD27 and CD70 superior to the comparison antibodies in this highly relevant bioassay. conclusions can be drawn.

Table 5: Chimeric Lama-Human CD7010 Tested in Raji-Based Co-Culture Analysis Neutralized Expressed as IC50 Value (ng/ml) of mAbs and Reference mAbs Repulsive Power mAb IC50 (ng/ml) 9D1 29 5F4 32 9B2 213 4D2 54 9E1 517 582 19 27B3 5 19G10 16 59D10 10 Example 7: Antil_<0ra-Related Cellular Cytotoxicity Efficacy Antibody-Induced Cellular Cytotoxicity (ADCC) McEarchem et al. (Blood (2007) 109: peripheral blood mononuclear cells (PBMC) heparinized whole with standard Ficoll separation It was purified from blood and used as a source of NK cells (ie, effector cells). A few The blood of an independent donor was used. Cells 200 U/ml human IL-2 (induction of NK cells 2x106 /ml was suspended in nutrient medium containing (for) and incubated at 37°C overnight.

The next day, adherent and non-adherent cells were collected and washed once in culture medium.

Target cells became and marked with Cr5 '. Target/effector cell ratio It was 1:20 and 2 hours in the presence of different concentrations of antibodies in the culture medium. incubation was performed. Chimeric llama-human mAbs and comparison antibodies are different was tested for ADCC activity on 786-0 cells at concentrations.

The percentage of lysis was determined by the following equation: Lysis % heat' = (sample CPM-spontcm sal Well CPM)/(max sal @1 CPM-Spontaneous raft @1 The results were achieved through ADCC activity of all chimeric llama-human mAbs. shows that they induce the lysis of cells (Figure 7). SIMPLE mAb 27B3 l ug/ml It has a power of around and therefore a comparable SGN70 affinity variant with lC50 is 10 times stronger than 9D]”; ADCC if comparison SGN70 In terms of comparison, it is 2.5 times more powerful than the MDX69A7. SIMPLE antibody 27B3 combines best-in-class receptor-ligand blocking power with superior ADCC potency and therefore, it has a positive therapeutic profile. Example 8: Complementary-Independent Cytotoxicity Potency of CD70,e-Specific mAbs Definer-Induced Cytotoxicity (CDC) properties of antibodies McEarchern et al. In the control experiment, the U cells were CD70 by FACS analysis. tested for the presence of antigen. In a later experiment, a chimeric lama-human CD70 mAb in 3 different concentrations and 3%, 6%, or 9% human serum (human supplemental Test for CDC activity on both cell lines in the presence of was done. U266 cells gave the best signal-to-noise ratio in the presence of 9% serum. concluded.

In a next experiment, U266 cells were grown in the absence or presence of 9% human serum. With chimeric llama-human mAbs (Figure 8, top panel) or reference CD70 at 0.001-20 ug/ml mAbs (Figure 8, lower panel) and incubated at 37°C for 2 hours. cells centrifuged and the number of dying cells was determined by adding Propidium Iodide. Cell lysis Measured in FACS. All chimeric llama-human mAbs in the presence of 9% human serum CDC activity was demonstrated on U266 cells. SIMPLE antibody 27B3 comparison SGN70 has a similar IC50, again the MDX69A7 is 10 times less powerful. Example 9: Potency of Antibody-Dependent Cellular Fagocytosis Analyzing the Antibody-Associated Cellular F-agocytosis (ADCP) properties of SIMPLE mAbs carried out. In this assay, human peripheral blood mononuclear cells (PBMC) are standard. It was purified from heparinized whole blood by Ficoll separation. Blood from different donors was used.

Cells were incubated overnight at 37°C in RPMF containing 10% FCS in a tissue culture flask. was done. Non-adherent cells were removed and adhered cells were added for 15 days with 500 U/ml rhGM-CSF (7.5 Culture was done in 75 ml of X-VIVO 15 medium containing µg/ 75ml). if you do it after 15 days cells were harvested (monocyte-derived macrophages (MDM). 8.0 x loaded with red dye PKH26 incubated with mAb. Then 2.0 X104 MDM cells were added (411 targets/effectors) ( and after 1 hour incubation at 37°C, the cells were centrifuged, resuspended in 100 µl of PBS 1% BSA and V-based 96 transferred to a well plate. Incubate the macrophages for 30 minutes by keeping the plates on ice. anti-CDI lb monoclonal antibody (FITC-conjugated) was added for staining, then cells were washed twice with PBS and fixed with 1% paraformaldehyde (in PBS). samples Scoring of phagocytosis in double-stained cells was analyzed by FACS. Results in Figure 9° are shown, and these results indicate that all chimeric llama-human mAbs tested have ADCPs. It shows that they are strong in terms of inducing. SIMPLE antibody 27B3 100 ng/ml Tumor labeled with macrophages at concentrations of 1 tig/ml with an IC50 of around induced phagocytosis in up to 50% of cells. Example 10: Internalization of Anti-CD70 mAb91s in Tumor Cell Line 786-O Binding of CD70 antibodies to cell line 786-O derived from renal carcinoma. Proven to cause rapid (within 1 hour) internalization of the antibody-receptor complex to test the internalization of antibodies and comparison mAbs, 786-0 cells were cultured in a 96-well microtiter plate and overnight at 37°C. incubated. 2.5 µg/ml mAb was added and incubated with cells for 0-24 hours at 37°C. plates Coded "INSIDE" representing the amount of mAb that is internalized) or PBS ("OUTSIDE" coded and represents the amount of mAb that binds to the receptor outside the cell). This Then, cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, with PBS. washed and incubated for 5 minutes with 02% Triton-X-100 ("INSIDE") or PBS ("OUTSIDE") was done. The cells were then washed twice and with 100 mM glycine for 10 minutes at room temperature. washed, followed by a 30-minute incubation with PBS+1% BSA. Finally, Li- Prior to analysis on the Cor Odyssey infrared scanner, cells were used for goat anti-human Fc (Jackson Stained with Immunoresearch.

The mean MFI of antibody remaining outside the cell, that is, internalized, is shown in Figure 10. It was rated as EXTERNAL. The results showed that some mAbs compared to other mAbs. shows that they stay longer on the surface (higher signal). With this, None of the mAbs are fully internalized: initially (between 0 and 2 hours) mAb The internalization goes very quickly, but 30-40% of the mAb can be incubated at 37°C for 24 hours. Even after this, a steady state remains outside the cell, as if it has been reached. SIMPLE10 antibody 27B3 internalizes at the same rate as the comparison antibodies SGN70 and MDX69A7.

SIMPLE antibodies of 9E1 and 19G10 as well as others after 24 hours of incubation It is interesting to observe that it remains at high levels even outside the cells (lower panel, Fig. ). ADCC is at high levels for effective induction of CDC and ADCP effects. Recruitment of effector immune cells responsible for cell death of CD70* specific antibody It is important to stay out of targeted cancer cells for this purpose. On the other hand, rapidly internalized Antibodies have potential as Antibody Drug Conjugates (ADCs). Example 11: In A Tumor Xenograft Model of Chimeric Lama-Human Mabies Vivo Activity 106 Raji cells C.B.-17 in 0.2 mL PBS to induce disseminated disease lateral tail of combined immunodeficiency (SCID) mice (Harlan, Indianapolis, IN) injected into his vein. After the injection, all the mice were collected and then the group 9 mice per each were randomly assigned to various treatment groups. antibodies intraperitoneal It was given as 0.5 ml by injection 1, 4, 8, 11, 15 days after cell implantation.

Mice were monitored at least twice a week and observed 15% to 20% weight loss, hunched posture, lethargy, killed when they showed signs of illness, including cranial swelling or dehydration. Mice tested. This variant of Fc-dead has been previously described in the literature and its ADCC and CDC activity and has much lower ADCP activity (McEarchem et al., Clin Cancer Res data show that the mean survival time for control mice was 27 days. 0.1mg/kg Mean survival time of at least 67 days when given at doses equal to or greater than 4lD12 elongated. However, a dose of 0.01 mg/kgt is effective. Potency of the Fc-dead variant of 4lD12 in vivo is absent, which indicates the importance of ADCC, CDC and ADCP in this model. Example 12: Expression of Non-fucosylated mAb31s Antibodies with low amounts of fucosyl residues increase ADCC by 10-1000 fold. CHO cell line Ms without fucosyltransferase gene A eukaryotic expression encoding the heavy and light chain of chimeric llama-human CD70 mAbs. was electroporated with the vector. Drug resistance clones Excel] 325-PF CHO nutrient selected with on-site growth. IgG production by a standard CD70 binding ELISA. scanned for.

Table 6: VH Sequence Obtained from CD709e Specific Llama 1C2 ELQVVESGGGLVQPGGSLRLSCAASGFTLS 1 NYWMH 10 9D] EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 8312 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 8012 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 9131 EVQLQESGGGVVQPGGSLRLSCAASGFTFD 3 DYAMS 12 534 EVQVQESGGGLVHPGGSLKLSCAASGFTFD 4 TYAMS 13 532 QVQLVESGGDLVQPGGSLRLSCAASGFTVS 5 NPAMS 14 6D5 EVQLVQPGAELRKPGASVKVSCKASGYTFT 6 SYYID 15 4D2 QVQLQESGPGLVKPSQTLSLACTVSGGSIT 7 TSYYYWS 16 9A] EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 SYAMS 17 9G2 QVQLVESGGGLMQPGGSLRLSCAASGFTFS 8 SSAMS 18 932 QLQVVESGGGLMQPGGSLRLSCAASGFTFS 9 GSAMS 19 2733 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 24E3 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 Fab FRAME 1 SERIES CDRl SERIES 13. 13. 33D8 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 24F2 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 VYYMN 11 2436 EVQLQESGGGVVQPGGSLRLSCAASGFTFD 3 DYAMS 12 19010 EVQLQESGGGVVQPGGSLRLSCAASGFTFD 3 DYAMS 12 45312 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 AYYMN 20 4539 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 AYYMN 20 45F8 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 AYYMN 20 45Ai2 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 AYYMN 20 4536 EVQLVESGGGLVQPGGSLRLSCAASGFTFS 2 AYYMN 20 1C2 WVRQAPRKGLEWVS 21 TISTDNSRTYYADSVKG 26 9D1 WVRQAPGKGLEWVS 22 DINNEGGTTYYADSVKG 27 8312 WVRQAPGKGLEWVS 22 DINNEGDTTYYADSVKG 28 8C12 WVRQAPGKGLEWVS 22 DINNEGDTTYYADSVKG 28 931 WVRQAPGKGLEWVS 22 SIYMYDSSTYYADSVKG 29 5F4 WVRQAPGKGLEWVS 22 AISWSGGETFYAESMKG 30 532 WVRQAPGKGLEWVS 22 EITNYGYNRYYADSVKG 31 6D5 WVRQAPGQGLEWMG 23 RIDPEDGGTKYAQKFQG 32 4D2 WIRQPPGKGLEWMG 24 AIGSRGSTYYSPSLKT 33 9A1 WVRQAPGKGLEWVS 22 DlNSGGGSTKYNDSVKG 34 902 WVRQAPGKGLEWVS 22 SIYSDSSYTYYADSVKS 35 9B2 WVRQAPGKGLEWVS 22 SIYSHSMYTYYADSVKS 36 2733 WVRQAPGKGLEWVS 22 DlNNEGGTTYYADSVKG 27 24E3 WVRQAPGKGLEWVS 22 DINNEGGTTYYADSVKG 27 33D8 WVRQAPGKGLEWVS 22 DINNEGGTTYYADSVKG 27 24F2 WVRQAPGKGLEWVS 22 DINNEGGTTYYADSVKG 27 24B6 WVRQAPGKGLEWVS 22 SIYMYDSSTYYADSVKG 29 i 9G I 0 WVRQAPGKGLEWVS 22 SIYMYDSSTYYADSVKG 29 45312 WVRQAPGKGLEWIS 25 DINNEGYETYYADSVKG 37 45D9 WVRQAPGKGLEWIS 25 DINNEGYETYYADSVKG 37 45F8 WVRQAPGKGLEWIS 25 DINNEGYETYYADSVKG 37 45A 1 2 WVRQAPGKGLEWIS 25 DINNEGYETYYADSVKG 37 45B6 WVRQAPGKGLEWIS 25 DINNEGYETYYADSVKG 37 59D10 WVRQAPGKGLEWVS 22 GISGVTGGSSTSYADSVKG 259 Fab FRAME 3 SERIES CDR3 SERIES RFtlSRDHAKNTLILQMNS lCZ LKSE DTAVYYCIR 38 GSDYEH 49 RFTi SR DNAKNTLTLOMN 9Dl SLKPE DTALYYCVR 39 DAGYSNHVPIFDS 50 R FTlSRDNAKNTLTLQM D 8B12 SLKPE DTALYYCVR 40 DAGYSNHVPIFDS 50 R FTlSR DNAKNTLTLQM D 8C 12 SLKPE DTALYYCVR 40 DAGYSNHVPIFDS 50 R FTlSTDNAKNTVYLQMN 9E1 SLKSE DTAVYYCAK 41 DINRSYGSWSHFGPIFSS 51 R FTlSRNNAKNTLYLQMN 5F4 SLKSE DTAVYYCAR 42 GMGLAEGLTD 52 RFTISTDNAKNTLYLQMNS 5B2 LRSEDSAVYYCTA 43 SLGLEYGYGD 53 RVTFTADASTSTAYVELSS 6D5 LRSEDTAVYYCAS 44 RRRDFDY 54 RTSISRDTSKNOFTLOLSS VTGEITYNSGSYYYTLNLFD 4D2 VTPEDTAVYYCAR 45 Y 55 RFAISRDNAKNTLYLQMN 9Al SLKPEDTAVYYCAK 46 EGGSGRYWTNEYDY 56 RFTISTDNAKNTLYLOMNS 9G2 LKPDDTAVYYCAG 47 SSDYEGSFAS 57 RFTISTDNAKNTLYLOMNS 9B2 LKPDDTAVYYCAA 342 SSDYEGLFVS 58 RFTl SR DNAKNTLTLOMN 27133 SLKPE DTALYYCVR 39 DAGYSNHVPIFDS 50 RFTl SRDNAKNTTLTLQMN 24E3 SLKPE DTALYYCVR 39 DAGYSNHVPIFDS 50 RFTI SRDNAKNTLTLQMN 33D8 SLKPEDTALYYCVR 39 DAGYSNHVPIFDS 50 RFTi SR DNAKNTLTLQMN 24F2 SLKPE DTALYYCVR 39 DAGYSNHVPIFDS 50 R FTlSTDNAKNTVYLOMN 24B6 SLKSE DTAVYYCAK 41 DINRSYGSWSHFGPIFSS 51 R FTlSTDNAKNTVYLQMN l9G10 SLKSE DTAVYYCAK 41 DINRSYGSWSHFGPIFSS 51 R FTlSRDNAKNTLTLQM D 45B12 SLKPEDTARYYCVR 48 DAGYSNHVQIFDS 59 R FTlSRDNAKNTLTLQM D 45D9 SLKPE DTARYYCVR 48 DAGY SN HVQIFDS 59 R FTlSRDNAKNTLTLOM D 45F8 SLKPE DTARYYCVR 48 DAGYSNHVQIFDS 59 Fab FRAME 3 SEQ ID. CDR3 SEQ ID› RFtlSRDNAKNTLTLQMD 45A I 2 SLKPEDTARYYCVR 48 DAGYSNHVQIFDS 59 R FTlSRDNAKNTLTLQM D 4536 SLKPE DTARYYCVR 48 DAGYSNHVQIFDS 59 RFTISRDNG KNTLYLOMN RFTiSRDNDKNTLYLQMN 59DI 0 SLIPEDTAVYYCAT 261 TSGTYYFIPEYEY 263 Fab FRAME 4 SEQ ID. 1 C2 WGQGTQVTVSS 60 9DI WGQGTQVIVAS 61 813 1 2 WGQGTQVIVAS 61 8C12 WGQGTQVIVAS 61 9E1 WGQGTQVTVSS 60 5F4 WGQGTQVTVSS 60 5132 WGQGTQVTVSS 60 6D5 WGQGTQVTVSS 60 4D2 WGQGTQVTVSS 60 9A i WGQGTQVTVSS 60 9G2 WGQGTQVTVSS 60 982 WGQGTQVTVSS 60 27B3 WGQGTQVIVAS 61 24E3 WGQGTQVIVAS 61 33D8 WGQGTQVIVAS 61 24F2 WGQGTQVIVAS 61 24B6 WGQGTQVTVSS 60 19010 WGQGTQVTVSS 60 45312 WGQGTQVIVAS 6i 45D9 WGQGTQVIVAS 61 45F8 WGQGTQVIVAS 61 45A 12 WGQGTQVIVAS 61 WGQGTQVIVAS 61 WGKGTLVTVSS 264 59DIO WGQGTQVTVSS 60 Table 7: VL Sequence Obtained from CD70,e-Specific Llama (Buv Colored O Amber DUR Coded by Codon) 1c2 QTVVTQEPSLSVSPGGTVTLTC 62 GLSSGSVTTTNYPG 77 931 QAVVTQEPSLSVSPGGTVTLTC 63 GLSSGSVTSSHYPG 78 8312 QTVVTQEPSLSVSPGGTVTLTC 62 GLSSGSVTSSNYPG 79 8C12 QAVVTQEPSLSVSPGGTVTLTC 63 GLTSGSVTSSNYPD 80 931 QAVVTQEPSLSVSPGGTVTLTC 63 GLTSGSVTSSNYPD 80 534 QAVVTHPPSLSASPGSSVRLTC 64 TLISGDNIGGYDIS 81 532 QSALTQPPSVSGTLGKTLTISC 65 AGTSSDVGYGNYVS 82 6D5 QSALTQPSAVSVSLGQTARITC 66 QGGNARFSSFA 83 432 QSVLTQPPSLSASPGSSVRLTC 67 TLSSGNSVGNYDIS 84 9A] QSALTQPSALSVTLGQTAKITC 68 QGGRLGSSYAH 85 9G2 QSVVTQPPSLSASPGSSVRLTC 69 TLSSGNSVGNYDIS 84 932 QAVLTQPPSLSASPGSSVRLTC 70 TLNSANSVGSYDIS 86 2733 QAVVTQEPSLTVSPGGTVTLTC 71 GLKSGSVTSTNFPT 87 2433 QAVVTQEPSLSVSPGGTVTLTC 63 GLTSGSVTSDNFPV 88 3338 QSALTQPSTVSVSLGQTARITC 72 RGDSLERYGTN 89 2432 QSALTQPSAVSVSLGQTARITC 66 RGDTLRNYHAN 90 2436 QPVLTQPSAVSVSLGQTARITC 73 QGGYYTH 91 19G10 NFMLTQPSAVSVSLGQTARITC 74 QGGYYTH 91 45312 QAVLTQPSSVSVSLGQTAKITC 75 QGGNLGLYGAN 92 4539 QAVLTQPSSVSVSLGQTAKITC 75 QGGNLWLYGAN 93 45F8 QAVLTQPSSVSVSLGQTANITC 76 QGGNLGLYGAN 92 45A12 QAVVTQEPSLSVSPGGTVTLTC 63 GLSSGSATSGNYPE 94 4536 QAVVTQEPSLSVSPGGTVTLTC 63 GLSSGSVTSSNYPD 95 1C2 WFQQTPGQAPRTLIY 96 STSSRHS 109 931 WYQQTPGQAPRLIF 97 NT'NSHRS 110 8312 WYQQTPGQAPRVLIY _98 NTNNRHS 111 8C 12 WYQQTPGQAPRLLIY 99 NTNSRHS 1 10 Fab Frame 2 SEQ ID. CDR2 SEQ ID. 9El WYQQTPGQAPRLLIY 99 NTNSRHS 1 10 5F4 WYQQKTGSPPRYLLY 100 YYSDSYKHQS S 1 12 5B2 WYQQLPGTAPKLLIY 101 RVSTRAS 1 13 6D5 WYQQKPGQAPVQVIY 102 YNTNRPS 1 14 4D2 WYQQKAGSPPRYLLY 103 YYSDSYKNQGS 1 15 9A1 WYQQKPGQAPVLVIY 104 GNNYRPS 1 16 9G2 WYQQKAGSPPRYLLY 103 YYSSDVKHQGS 117 9B2 WYQQKAGSPPRYLLY 103 YYSDSLSHQGS 118 27B3 WYQQTPGQAPRLLIY 99 NTNTRHS 1 19 33D8 WYQQKPGQAPVLVIY 104 DDDSRPS 121 24F2 WYRQKPGQAPVLVIY 105 GDDIRPS 122 24B6 WYQQKPGQAPVLVIY 104 INNNRPS 123 45D9 WYQQNPGRAPILLIY 106 GDNQRPL 126 45F 8 WYQQNPGRAPILLF Y 107 GDNYMPL 127 45 B6 WYQQTPGQAPRLLIY 99 NTNSRHS 1 10 Fab Frame 3 SEQ ID. CDR3 SEQ ID.

GVPSRFSGSISGNKAALTITGAOP 1C2 EDEADYYC 129 ALEEIGSYTYM 148 VP RF | NKAALTIT A P 9D 1 SDEIDYSSS SG G 0 129 ALLNIDDGSTM 149 GVPS RYSGSISGN KAALTITGAE P 8B12 EDEADYYC 130 NLHLGSYTPM 150 GVPSRFSGSISGNKAALTITGAOP 8C12 EDEADYYC 131 ALYWGYGTNVDV 151 GVPSRFSGSISGNKAALTITGAOP GVPSRFSGSKDASANAG LLLISGL 5F4 OSEDEADYYC 132 SAYKSGSYKAPV 153 GMPDRFSGSKSGNTASLTISG LQ 5B2 SEDEADYYC 133 ASYTTNNKPV 154 GIPARFSGSSSGGAATLTISGAQA GVPSRFSGSKDPSANAGLLLISGL 4D2 QAEEDEADYYC 135 SVSNSGTYKPV 156 Fab Frame 3 SEQ ID. CDR3 SEQ ID.

GIPERFSGSSSGDTATLTISGAQA 9A1 EDEAVYYC 136 QSGSSNTNVM 157 GVPSRFSGSSDASANAGLLLISGL 9G2 QPEDEADYYC 137 SAYKSGSHV 158 GVPSRFSGSTDASANAGLLLISGL 9B2 OPEDEADYYC 138 SAYNRGSHV 159 GVPSRFSGSISENKAALTlTGAQP 27B3 EDEAEYFC 139 ALFISNPSVE 160 GVPSRFSGSITGNKAILTITGAQPE GIPERFSGSSSGATAALTISGAOA 33D8 EDEGDYYC 141 QSADSSGNAV 162 24F2 EDEADYSYGCS GG SG O 142 QSSDSSGYRVV 163 GIPERFSGSISGNTATLTISGAOVE 24B6 DEADYYC 143 QSGSSSSTIPV 164 GIPERFSGSSSGNTATLTISGAOA GIPERFTISKSGGTATLTIDGAOAE GIPERFTISKSGGTATLTIDGAOAE 45D9 DESDYYC 145 QSADYSGNSV 165 GVPSRFSGSISGNKAALTITGAOP GVPSRFSGSISGNKAALTITGAOP 57B6 EDEADYYC 129 ALYMGSGSANAM 271 GIADRFSGSKAGNTASLTISGLQS Fab Frame 4 SEQ ID. 1 C2 FGGGTHLTVLG 169 9D 1 FGGGTHLTVLG 169 8B12 FGGGTKLTVLG 170 8C12 FGGGTKLTVLG '170 9E1 FGGGTKLTVLG 170 SP 4 F GGTTHLTVLG 169 5B2 F GGTTHLTVLG 169 6D5 FGGGTTLTVLG 171 4D2 FGGGSKLTVLG 172 9A1 FGGGTHLTVLS 173 9G2 FGGGTKLTVLG 170 Fab Frame 4 SEQ ID. 9B2 FGGGTKLTVLG 170 27B3 FGGGTQLTVLS 174 24E3 FGGGTRLTVLG `175 33D8 FGGGTHLTVLG 169 24F 2F GGGTKLTVLG 170 24B6 F GGGTKLTVLG 170 19G10 FGGGTKLTVLG 170 45B12 FGGGTKLTVLG 170 45D9 FGGGTKLTVLG 170 45F8 FGGGTKLTVLG 170 45A12 FGGGTKLTVLG 170 45B6 FGGGTELTVLG 176 57B6 FGGGTHLTVLG 169 59D10 FGGGTHLTVLG 169 Table 8: Whole Bov VH from Llama Full Length VH Domain Array ELQVVESGGGLVQPGGSLRLSCAASGFTLSNYWMHWVRQAPRKG LEWVSTISTDNSRTYYADSVKGRFFISRDHAKNTLILQMNSLKSEDT AVYYCIRGS DYEHWGOGTOVTVSS EVQLVESGGGLVOPGGSLRLSCAASGFTFSVYYMNWVROAPG KG LEWVSDINN EGGTTYYADSVKGRFTISR DNAKNTLTLOMNSLKPED TALYYCVRDAGYSNHVPIFDSWG OGTQVIVAS EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPG KG LEWVSDINN EG DTTYYADSVKGRFTISR DNAKNTLTLOMDSLK PED TALYYCVRDAGYSNHVPIFDSWGQGTOVIVAS EVOLVESGGGLVO PGG SLR LSCAASGFTFSVYYM NWVROAPG KG LEWVSDINN EG DTTYYADSVKGRFTISR DNAKNTLTLOMDSLK PED TALYYCVRDAGYSNHVPIFDSWGOGTQVIVAS EVOLOESGGGVVQPGGSLRLSCAASGFTFDDYAMSWVROAPGKG LEWVSSIYMYDSSTYYADSVKG RFTISTDNAKNTVYLOMNSLKSED TAVYYCAKDIN RSYGSSWSH FG PIFSSWGOGTOVTVSS EVOVOESGGGLVHPGGSLKLSCAASGFTFDTYAMSWVRQAPGKG LEWVSAISWSGG ETFYAESMKGRFTISHN NAKNTLY LOM NS LKSED TAVYYCARGMGLAEGLTDWGQGTOVTVSS QVQLVESGGDLVOPGGSLRLSCAASG FTVSN PAMSWVROAPG KG LEWVSE[TNYGYNRYYADSVKGRFTlSTDNAKNTLYLQMNSLRSED SAVYYCTASLGLEYGYG DWGOGTOVTVSS EVQLVQPGAELRKPGASVKVSCKASGYTFTSYYl DWVROAPG OGL EWMG RlDPEDGGTKYAQKFOGHVTFTADASTSTAYVELSSLRSED TAVYYCASRRRDFDYWGQGTQVTVSS QVQLOESG PGLVKPSQTLSLACTVSGGSITTSYYYWSWIROP PGK GLEW MGAlGSRGSTYYSPSLKTRTSISRDTSKNOFTLOLSSVTPED TAVYYCARVTG EITYNSGSYYYT LN LF DYWGOGTQVTVSS Full Length VH Domain Array EVQLVESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKG LEWVSDl NSGGGSTKYNDSVKG R FAISR DNAKNTLYLQM NS LKF'ED TAVYYCAKEGGSG RYWTN EYDYWGQGTOVTVSS QVQLVESGGGLMQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKG LEWVSSIYSDSSYTYYADSVKSRFTlSTDNAKNTLYLOMNSLKPDDT AVYYCAGSSDYEGSFASWGOGTOVTVSS OLOVVESGGGLMOPGGSLRLSCAASG FTFSGSAMSWVRQAPGK GLEWVSSIYSHSMYTYYADSVKSRFTISTDNAKNTLYLQMNSLKPD DTAVYYCAASSDYEGLFVSWGOGTOVTVSS EVQLVESGGGLVOPGGSLRLSCAASGFTFSVYYMNWVROAPGKG LEWVSDINN EGGTTYYADSVKGRFTISH DNAKNTLTLOMNSLKPED TALYYCVRDAGYSNHVPIFDSWG OGTQVIVAS 24133 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVROAPGKG LEWVSDINN EGGTTYYADSVKGRFTISRDNAKNTLTLOMNSLKPED TALYYCVRDAGYSNHVPIFDSWGOGTOVIVAS EVOLVESGGGLVOPGGSLRLSCAASGFTFSVYYMNWVROAPG KG LEWVSDINN EGTTTYYADSVKGR FTISH DNAKNTLTLOMNSLKPED TALYYCVRDAGYSNHVPIFDSWGOGTQVIVAS EVOLVESGGGLVOPGGSLHLSCAASGFTFSVYYMNWVROAPG KG LEWVSDINN EGGTTYYADSVKGRFTISRDNAKNTLTLOMNSLKPED TALYYCVRDAGYSNHVPIFDSWG OGTQVIVAS EVQLOESGGGVVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKG LEWVSSIYMYDSSTYYADSVKG RFTISTDNAKNTVYLOMNSLKSED TAVYYCAKDIN RSYGSSWSH FG PIFSSWGOGTQVTVSS 19G10 EVQLOESGGGVVOPGGSLRLSCAASGFTFDDYAMSWVRQAPGKG LEWVSSIYMYDSSTYYADSVKG RFTISTDNAKNTVYLOMNSLKSED TAVYYCAKDIN RSYGSSWSH FG PIFSSWGOGTOVTVSS 45812 EVOLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVROAPG KG LEW ISDI NN EGYETYYADSVKG RFTISRDNAKNTLTLOM DSLKPE DT ARYYCVRDAGYSNHVOIFDSWGOGTOVIVAS EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPG KG LEW ISDl NN EGYETYYADSVKG RFTISRDNAKNTLTLOM DSLKPE DT ARYYCVRDAGYSNHVOIFDSWGOGTQVIVAS EVOLVESGGG LVOPGGSLRLSCAASGFTFSAYYMNWVROAPG KG LEW lSDl NNEGYETYYADSVKG RFTISRDNAKNTLTLQM DSLKPE DT ARYYCVRDAGYSNHVOIFDSWGOGTOVIVAS 45A12 EVQLVESGGGLVOPGGSLRLSCAASGFTFSAYYMNWVROAPG KG LEWlSDINNEGYETYYADSVKGRFTISRDNAKNTLTLOM DSLKPE DT ARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS EVQLVESGGG LVOPGGSLRLSCAASG FTFSAYYMNWVROAPG KG LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLOMDSLKPE DT ARYYCVRDAGYSNHVOIFDSWGOGTOVIVAS QLOLVESGGG LVOPGGSLRLSCAASGFSFSHYAMSWVROAPGKG LEWVSGDNTYDGGTRYODSVKGRFTISRDNGKNTLYLQMNSLKPE DTAVYYCAKDTGRGIMGEYG MDYWG KGTLVTVSS 59D10 EVQLVESGGGLVQPGGSLRLSCAASELS FSlSEMTWVRQAPGKGL EWVSGISGVTGGSSTSYADSVKGR FTIS R DNDKNTLYLOMNSLIPE DTAVYYCATTSGTYYF I PEYEYWGOGTOVTVSS Table 9: VL Obtained from Full-Length Llama (Q Amber DUR Codon with Burrow Color) Coded by Full Length Series Full Length Series QTVVTQE PSLSVSPGGTVTLTCG LSSGSVTTTNYPGWFOOTPGQA PRTLIYSTSSRHSGVPSR FSGSISGNKAALTITGAOPEDEADYYCAL EEIGSYTYMFGGGGTHLTVLG QAVVTOE PSLSVSPGGTVTLTCGLSSGSVTSSHYPGWYQQTPGO APR LLIFNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCA LLNIDDGSTMFGGGGTHLTVLG QTVVTOEPSLSVSPGGTVTLTCGLSSG SVTSSNYPGWYOOTPGQA PRVLIYNTNN RHSGVPSRYSGSISGNKAALTITGAE PEDEADYYCNL HLGSYTPMFGGGTKLTVLG OAVVTQEPSLSVSPGGTVTLTCG LTSGSVTSSNYPDWYOOTPGOA PRLLIYNTNSRHSGVPSRFSGSISGNKAALTITGAOPE DEADYYCAL YWGYGTNVDVFGG GTKLTVLG QAVVTQE PSLSVS PGGTVTLTCG LTSGSVTSSNY P DWYQOT PGOA PRLLlYNTNSRHSGVPSRFSGSISGNKAALTITGAOPE DEADYYCNL YMG SGGSKVFGGGTKLTVLG QAVVTH PPSLSASPGSSVRLTCTLISG DN IGGYDISWYOOKTGSPP RYLLYYYSDSYKHOSSGVPSRFSGSKDASANAGLLLISG LOSEDEA DYYCSAYKSGSYKAPVFGGGGTHLTVLG QSALTOPPSVSGTLGKTLTISCAGTSSDVGYG NYVSWYOOLPGTA PKLLlYRVSTRASGMPDRFSGSKSGNTASLTlSGLQSEDEADYYCA SYTTNNKPVFGGGGTHLTVLG OSALTOPSAVSVSLGOTAR lTCOGGNARFSSFAWYOOKPGOAPV QVIYYNTNRPSG I PARFSGSSSGGAATLTISGAQAE DEADYYCQSY ESGNYVFGGGTTLTVLG OSVLTOPPSLSASPGSSVRLTCTTLSSGNSVG NYDlSWYOQKAG SP PRYLLYYYSDSYKNOGSGVPSRFSGSKDPSANAGLLLISGLOAEDE ADYYCSVSNSGTYKPVFGGGSKLTVLG OSALTOPSALSVTLGOTAKITCOGGRLGSSYAHWYQQKPGOAPVL VlYGN NYRPSGl PERFSGSSSG DTATLTISGAQAED EAVYYCQSGS SNTNVMFGGGGTHLTVLS QSVVTOPPSLSASPGSSVRLTCTTLSSGNSVGNYDISWYOQKAGSP PRYLLYYYSDSVKHQGSGVPSRFSGSSDASANAGLLLISGLQPEDE ADYYCSAYKSGSHVFGGGTKLTVLG OAVLTOPPSLSASPGSSVF1LTCTLNSANSVGSYDISWYOQKAGSP PRYLLYYYSDSLSHOGSGVPSRFSGSTDASANAGLLLISGLOPEDE ADYYCSAYN RGSHVFGGGTKLTVLG QAVVTQE PSLTVS PGGTVTLTCGLKSG SVTSTNFPTWYOQTPGOA PRLLIYNTNTRHSGVPSRFSGSISENKAALTITGAOPEDEAEYFCAL FISNPSVEFGGGTOLTVLS 24133 QAVVTOEPSLSVSPGGTVTLTCGLTSGSVTSDN FPVWYOOTPGQA PRLLlYTl NSRHSGVPSRFSGSITGNKAI LTITGAOPEDEADYYCALY LEN FAN EFGGGTRLTVLG OSALTOPSTVSVSLGOTARITCRGDSLERYGTNWYQQKPGQAPVL VIYDDDSR PSGIPERFSGSSSGATAALTISGAQAEDEG DYYCOSAD SSGNAVFGGGGTHLTVLG OSALTOPSAVSVSLGOTARlTCRGDTLRNYHANWYROKPGOAPVL VlYGDDIRPSGlPERFSGSRLGGTATLTVSGAOAEDEADYYCOSSD SSGYRVVFGGGTKLTVLG QPVLTOPSAVSVSLGQTAR ITCQG GYYTHWYQQKPGOAPVLVIYIN NNHPSGIPERFSGSSISG NTATLTlSGAOVEDEADYYCQSGSSSTIPV FGGGGTKLTVLG 19G10 NFMLTQPSAVSVSLGOTARlTCOGGYYTHWYOOKPGOAPVLVIYV NNNR PSGlPERFSGSSSGNTATLTISGAQAEDEAAYYCQSGSSSTI PVFGGGTKLTVLG Fab Full Length Series SERIES ID.

QAVLTOPSSSVSVSLGOTAKITCOGGNLGLYGANWYQONPGRAPIL LIYGDNYRPLGlPERFTISKSGGTATLTIDGAOAEDESDYYCOSADY 45812 SGNSVFGGGTKLTVLG 207 OAVLTQPSSVSVSLGQTAKITCQGGNLWLYGANWYQONPGRAPIL LIYGDNORPLGIPERFTISKSGGTATLTlDGAOAEDESDYYCOSADY 45D9 SGNSVFGGGTKLTVLG 203 QAVLTOPSSSVSVSLGOTANlTCOGGNLGLYGANWYOONPGRAPIL LFYGDNYMPLGIPERFTlSKSGGTATLTIDGAQAEN ESDYYCOSSDY 45F8 PGNSVFGGGTKLTVLG 209 OAVVTQEPSLSVSPGGTVTLTCG LSSGSATSGNYPEWYOOTPGQ APRLl lYNTASRHSGVPGR FSGSlSGNKAALTITGAQPEDEADYYCL 45A12 LYMGGSDFNFVFGGGTKLTVLG 210 QAVVTQEPSLSVSPGGTVTLTCGLSSGSVTSSNYPDWYQOTPGQA PRLLlYNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCAL 45B6 YMGSGSNNVVFGGGTELTVLG 21 1 OTVVTQEPSLSVSPGGTVTLTCGLKSGSVTSSNYPAWYOOTPGOA PRLLlYNTNSRHSGVPSRFSGSISGNKAALTITGAOPEDEADYYCAL OSVLTQPPSVSGSPGKTVTISCAGTSSDVG YGYYVSWYQOFPG MA PKLLIYDVNKRASG lADFl FSGSKAG NTASLTISG LOSEDEADYYCAS 59D10 YRSSANAVFGGGGTHLTVLG 277 Example 13: Anti-CD70 mAb 27B3 Germline Processing mAb 27B3 has the same canonical folding structure as the VH and VL regions and the highest amino Human germline gene with known fragments of human germline genes with acid sequence identity identified by comparison with their sequences. The human VH and VL regions closest to mAb 27 B3° human VH3-48 (% respectively. Most The proximal human germline JH and JL human germline gene cross-section sequences were JHS and JL7, respectively. Sequence with the closest human germline VH and VL sequences of the mAb 27B3 VH and VL regions alignment is shown in Figure 123. SIMPLE antibody 2783 and human germline Comparison of the V regions of the sequences 12 in the VH region and 13 in the VL region (CDR1, de 2 and 1) in CDR25 identified candidate humanizing mutations. in the VH and VL sequences of 27B3 necessary to accommodate humanizing mutations and the accompanying diversity. A general description of library sizes is given in Tables 10 and II.

Table 10: Mutations Targeted in the 27B3 VH Amino Acid Sequence All Possible Mutants Indicates Library Size Required to Cover Mutations Found in the Library Kabat Devegil aa Human Germline Human Germline Human Germline Probability Position aa Option l aa Option 2 aa Option 3 46 E V 050 Mutations Found in the Library Kabat Devegil aa Human Germline Human Germline Human Germline Probability Position aa Option l aa Option 2 aa Option 3 74 A S 0.50 77 T S 0.50 79 T Y 0.50 83/84 KP KT RA 0.33 89 L V 0.50 93/94 AR AK VR 0.33 108 Q L T M 0.25 110 1 0.50 112 AS 0.50 Table 11: Mutations Targeted in the 27B3 VL Amino Acid Sequence All Possible Mutants Indicates the Library Size Required to Cover. * in CDR] or CDR2 Regions Shows Mutations.

Kabat Devegil aa Human Germline Human Germline Human Germline Probability Position aa Option 1 aa Option 2 aa Option 3 2 A T 0.50 1 1-12 LT FS 0.50 26* K T 0.50 * TD 0.50 46 L T 0.50 53* T S 0.50 60 S D 0.50 61 R C 0.50 67/68 SE LG 0.50 87 FY 0.50 Germ encompassing all humanizing mutation combinations of VH_27B3 and VL_27B3 created by PCR-based assembly of the line-processed libraries (see Oligonucleotides overlapping with mutations were combined by PCR. Library with high affinity When the wild-type llama residue is critical for binding, the binding is completely both human and llama amino acids in every position where the mutation occurs to prevent its loss. approximately 1 x 10lo clones and the VL library contained approximately 8 x 10 9 clones. VH and VL libraries were combined and 1 x 1010 clones suitable for phage display screening (full size F ab plugin 91% ) reformatted as a single F ab library. Table 10 and lee required diversity primary heavy chain and light chain libraries, but the combined Fab library also contains all possible was large enough to contain the permutations (1.89 x 107). Example 14: Germline Processed 27B3 Fabs with High Human FR Identity Selecting Germline processing with both a high attenuation for CD70 and high human FR identity Phage display was used in the selection of the made 27B3 Fab*. specify as tain If necessary, Flag-TNC-CD70 was biotinylated and different amounts of Fab at various concentrations. incubated with expressing phage. Then phage and Flag-TNC-CD70 complexes was captured in a streptavidin-coated microtitration plate and biotinylated F lag-TNC- It was washed with CD70 at 37°C. The phage was separated with Trypsin and in the infection of TG1 cells used. Five selection rounds were carried out in each round, increasing the difficulty of the washing conditions.

Details of the conditions used in each selection round are given in Table 12.

Table 12: Selection Conditions for Germline Processed 27B3 Fabs Type Phage Input (nl) Antigen [pM] Wash Time Wash Temperature Washer Antigen In the 3rd, 4th and 5th rounds of selection, the eluted phage was transfected into TG1 and plated as a clone; individual clones were randomly selected for reanalysis. Periplasmic containing F abls fractions were prepared from IPTG-induced small-scale cultures. The dissociation rate of each Fab* is a It was determined using a CD70 coated CMS chip in the Biacore binding assay. 27B3`s Clones with Fab with similar divergence rates were sequenced. Over 94% of total people Segregation rates for clones with FR sequence identity, human FR of individual clones Its identity (VH, VL and total) is given in Table 13, The complete amino acid sequences of the Fab clones sequenced are given in Tables 14 and 15% and the CDR of 27B39. amino acid sequence variants are given in Table 161. Examination of these sequences reveals that the light chain extra (non-human) in the primary region of cyclic amino acid 29 of framework 1 indicates that it has mutations (A instead of T). 35G3, 40F1 and 39C3 clones variants (clones 53Cl, 53B1 and 53EI, respectively) have been made, corresponding to position 2 line has been done.

Table 13: More than 947% Total Human FR Identity with Germ Line Transactions Performed Decomposition Rate of 27B3 Fab Clones FR for Fab VL FR for VH Total FR isolation Sep. speed % of Clone Identity % Identity % Identity % Identity % Frequency [10454] Table 14A: Germline-treated 27B3 clones with greater than 947% total human identity with FR and CDR disrupted sequence identifiers VH amino acid sequences. Humanizing mutations are pale, primer-attached mutations are dark.

HEAVY FRAME i CDR] FRAME 2 27B3 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

DIZI ID. NO:2 NO:22 36A9 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:2 NO:22 53F1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. No:11 WVRQAPGKGLEWVS Sequence ID.

SEQ ID. No:2 NO:22 36D6 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:2 NO:22 53Gl EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:2 NO:22 3503 EYQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:2 NO:22 53C1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:2 NO:22 35F6 EVQLVESGGGLVQPGGSLRLSCASSGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:274 NO:22 36G2 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:2 NO:22 39D5 EVQLVESGGGLVQPGGSLRLSCAASGFTFS GYYMN SEQ ID. No:248 WVRQAPGKGLEWVS SEQ ID› DIZI IDA No:2 NO:22 42D12 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:2 NO:22 3501 EVQLVESGGGLVQPGGSVRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID. 41D12 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

DIZI IDV No:2 NO:22 41H8 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:2 NO:22 3502 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID. 40F1 EyQEVESGGGLVQPGGSLRLSCAASGFTFs VYYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:2 NO:22 5331 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:2 NO:22 3903 EVQLVASGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:275 NO:22 53151 EVQLVASGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

SEQ ID. No:275 NO:22 53H1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID› SEQ ID. No:2 NO:22 53A2 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYMN SEQ ID. NO:11 WVRQAPGKGLEWVS SEQ ID.

CHAIN 27133 DINNEGGTTYYADSVKG SEQ ID. NO:27 RFTISRDNAKNTLTLQMNSLKPEDTALYYCVR DAGYSNHVPIFDS WGQGTQVIVAS 36A9 DINNEGGTTYYADSVKG SEQ ID. NO:27 REUPRDHSKIITLTLQNNSLKPEDTAWYCVR DAGYSNHVPIFDS @GQGTQVIVSS 53F1 DINNEGGTTYYADSVKG SEQ ID. NO:27 RETISRDNAKNTLTLQMUSLKPEDTAWYCVR DAGYSNHVPIFDS Wçqsmvms 36D6 DINNEGGTTYYADSVKG SEQ ID. NO:27 R??IFRDIIAKIJSLYLQMIBLKPEDTALYW DAGYSNHVPIFDS V'FGQGTTIVWSS 53G1 DINNEGGTTYYADSVKG SEQ ID. NO:27 REUSRDNAKUTLYLQNNSLKPEDTAVYYCVR DAGYSNHVPIFDS WQGI'WIWIS 3503 DINNEGGTTYYADSVKG SEQ ID. NO:27 RET.I.SRDN'êKusLTLQMHSLKPEDTAWYCAR DAGYSNHVPIFDS Wçqsnamss 53Cl DINNEGGTTYYADSVKG SEQ ID. NO:27 RETISRDN'SKI'SLTLQNHSLKPEDTAWYCAR DAGYSNHVPIFDS WGQGTLVIWG 35F6 DINNEGGTTYYADSVKG SEQ ID. NO:27 REUSRDNSKUTLYLQNNSLRAEDTAVYYCVR DAGYSNHVPIFDS V'EÇQGITX7îR7AS 39D5 DINNEGGTTYYADSVKG SEQ ID. NO:27 asagoumqmmimsmmmm DAGYSNHVPIFDS Wçqsmrwss 42D12 DINNEGGTTYYADSVKG SEQ ID. NO:27 RETISRDIISKUTLYLQBINGLRAEDTAVYYCVR DAGYSNHVPIFDS V'EFQGIQY'T'VGS 35Gl DINNEGGTTYYADSVKG SEQ ID. NO:27 RET;SRDNAKQTLYLQ›INSLKPEDTAWYCAR DAGYSNHVPIFDS WEQGITLWAS 411312 DINNEGGTTYYADSVKG SEQ ID. NO:27 RET1_SP-DNSWSLYLQNI'SLRAEDTA""'YCAR DAGYSNHVPIFDS V'UPQGIEVTVGS 41H8 DINNEGGTTYYADSVKG SEQ ID. NO:27 RFTISRDNGKUTLYLQBIHSLRAEDTAVYYCAR DAGYSNHVPIFDS WGQGTQVWAS 35G2 DINNEGGTTYYADSVKG SEQ ID. NO:27 Rsr;;mummg»nimpgmmym DAGYSNHVPIFDS @QGTMVZSS 40F1 DINNEGGATYYADSVKG SEQ ID. No:249 RETFSvalsKljsLYLQMNSLRAEDTAVYYCAR DAGYSNHVPIFDS i-:çggmmss 53Bi DINNEGGATYYADSVKG SEQ ID. No:249 RSTAIISRDIIISIKNSLYLQMUSLRAEDTAVYYCAR DAGYSNHVPIFDS ç-;çggmvms 39C3 DINNEGGTTYYADSVKG SEQ ID. NO:27 RülêRDHSKI'TLYLQHHSLRAEDTAWYCAR DAGYSNHVPIFDS SEQ ID, NO: 286 SEQ ID N050 5351 DINNEGGTTYYADSVKG SEQ ID. NO:27 RsrpmisKurmgmismsmmm DAGYSNHVPIFDS . 53H1 DINNEGGTTYYADSVKG SEQ ID. NO:27 RÜIFRWSKI'SLYLQWLRREDTAVHCAR DAGYSNHVPIFDS Wcessmîviw 53A2 DINNEGGTTYYADSVKG SEQ ID. NO:27 RFTISFDNSKusLYLQNNSLMEDTAWYCAR DAGYSNHVPIFDS WGQGTIVWS SEQ ID, NO: 285 DIZI ID. NO:50 Table 14B: For complete VH amino acid sequences, with sequence identifiers, 94% “[maximum total VH amino acid sequences of germline-treated 27B3 clones with human identity SERIES HEAVY FULL VH SERIES 178 27B3 EL'QLVESGGGLVQPGGSLRLSCAASGFTFSVYYHHWVRQAPGKGLEWVSDII'vlllEGGTTYYADSVKG EFTI SRDIJAKI ITLQMIISLI( PEDTALYYCVRDAGYSUHVP IFDSWGQGTQV I'GAS 212 3 6A9 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMIIWVRQAPGKGLEWVSDI I'IIJEIGGTTY YÂDSVKG 213 5 3 F1 EX'QLVESGGGLVQPGGSLRLSCAASGFTFSVYYMIIWVRQAPGKGLE'A ISDI I'IIJEGGTTY YADSVKG RFTI SRDI'JÄKNTLTLQD'II ISLKPEDTPNYYCVRDPßYSlTHVPIFDSWGQGTQVIVÃS 214 3 ASS E1\-"QLVE3GGGLVQPGGSLRLSCAASGFSVYY[VIIIWVRQAPGKGLE'ATVSDII-llJEGGTTY YADSVKG RFTISRDNAKI ITLYLQMIISLKPEDTALYYCVRDAGY SIIHVP IFDSWGQGTIVT !5.3 215 53G1 EUQLVESGGGLVQPGGSLRLSCAASGFTFSVY`i'MlIWJRQAPGKGLE'ATUSDIlllJEGGITYYADSVKG RFII SRDI'JAKI'ITLYLQMI ISLKPEDTAYYYCVRDAGYSlIHVPIFDSWGQGTEIVIVSS 216 3 5G3 BVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYl'IIIEFNRQAPGKGLE'JWSDIIllJEGGTTY YADSVKG RFTISRDNSKIISLTLQMIISLKPEDTAVYY CARDAGYSIIHVPIFDSWGQGTLVTVS S 217 53C1 B`r'QLVE3GGGLVQPGGSLRLSCAASGFTFS'JYYl“lllüIRQAPGKGLEWVSDIHIJEGGTTY YADSVKG RFTISRDNSKHSLTLQMIISLI'ÄPBDTAVYYCARDAGYSIIl-lVPIFDSWGQGTL\7_'I\›'SS 218 3 5F6 BVQLVESGGGLVQPGGSLRLSCASSGFTE`SVYY[VIIIWVRQAPGKGLEW SDIHIJEGGTTYYADSVKG RE'T I 5 RDN'S KHTLYLQMNS LRLÃLBDTAVYYCVRDAGYS [IHVP IFDSWGQGTTVIVA S 219 3 6G2 EE'QLVESGGGLVQPGGSLRLSCAASGFTFSVYYMI'vIWJRQAPGKGLEWVSDII-IllEGGTTY YADSVKG RFTI SRDI'JAKI'ISLTLQMIISLRjAEDTAVYYCVRDAGYSNHVPIFDSWGQGTQVTVSS 220 39135 Eîi'QLVESGGGLVQPGGSLRLSCAAC GFTFSGYYl»ll']1'u"JRQAPG1(GLEW SDIl-JNEGGTTYYADSVKG RFT I S RDNAKNTLYLQMIISLR'ABDTAVYYCVRDAGYS I-IHVP IFDSWGQGTTVTVAS 221 42D12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMIIWVRQAPGKGLEWVSDIIlEIGGTTY YADSVKG RFTI SRDIJSKNTLYLQMIISLRÄEDTAVYYCVRDAGYSNHVPIFDSWGQGTQVTVSS 222 3 5G1 BVQLVESGGGLVQPGGSVRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDIlJIJEGGTTYÄDSVKG RFT I SRDIJAKNTLYLQMI'IS LKPBDTAVYYCARDPSYSUHVPIFDSWGQGTLVT-VAS 223 41D12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMHWVRQAPGKGLEWVSDI I-HJEGGTTY YADSVKG RFTI SRDN'SKUSLXLQMI ISLRAEDTAVYYCÄRDAGYSlTHVPIFDSWGQGTLVIVS S 224 41H8 E3\-"QLVECGGGLVQPGGSLRLSCAASGFTFSVYY[VIIIWVRQAPGKGLEWVSDIllJEGGITY YADSVKG RFTISRDNSKI ITLYLQMIISLRAEDTAVYY CARDAGY .SlIHVPIFDCWGQGTQVTVAS RFTISRDNAKNTLILQMIISLKPEDTAYYYCARDAGYSIIIHVPIFDSWGQGTLVIVSS RFT I SRDIISKNSLYLQMI IS LRAEIDTAVYY CARDAGY SNHVP IFDEWGQGTQVTVS S 227 5313] Bî-'QLVESGGGLVQPGGSLRLSCAASGFTFSVYY[~'llll^NRQAPGl RE'T I 5 RDNS KNSL-YL QMIJS LRABDTAVYYCÄRDAGYS [vIHVP IFDSWGQGTQVTVS S 228 3 9c3 EVQLVASGGGLVQPGGSLRLSCAASGFTFSVYYMI'WNRQAPGKGLEWVSDIl-IIJEZGGTTYYADSVKG RFT I 5 RDN'SKl-JTLYLQMIIS LRAEDTAVYY CARDAGYS [JHVP IFDEEVGQGTTuTVS S 229 53151 EVQLVASGGGLVQPGGSLRLSCAASGFTFSVYYMI'JWX-'RQAPGKGLEWVSDINIJEGGTTYYADSVKG 223 53H1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMIll”f\.”RQAPGKGLElru`\.”SDIHNEGGTTY YADSVKG RFTI SRDNSKNSLYLQMI ISIRÄEIDTAVYYCARDAGYSI'THVPIFDSWGQGTLVT-VSS 223 53A2 E\-"QLVESGGGLVQPGGSLRLSCAASGFTFSVYYIVII'IWVRQAPGKGLEIWVSDII-UJEGGTTY YZ-.DSVKG RFTISRDNSKIISLYLQNIISLRAEDTAVYYCARDAGYSUHVPIFDSWGQGTLYTVS S Table 15A: More than 94% total people with FR and CDR-distorted sequence identifiers VH amino acid sequences of identically germline engineered 27B3 clones.

Humanizing mutations are pale. Mutations with nrimer suffixes are in the same color as the buff.

LIGHT FRAME 1 CM! FRAME 2 1:," ›-. ç:r.-I-i'çcpi'.r5':: :ax-sut.- -3yipat'nrmirpr werWQEPEILW at i @sasîtairsr:vspxnntc ;'.ssx'nm ini* m-;qncouaui i' 3505 tanju& row :omur: :113:: 'nc 'n IFPT çrwiimmm' I. m tir-"ri'iccp-:uzmez-.run r: :3 't'n'n'mnr ::s-:mami: RLLTY sc-1; 3 magma:matem-nn:: 6.1.›:772'4'7: :um rqr-;qrmmnuu :.: : 01'::r;i:i~:.r:;'.~:iusI-›JI L:: i'..'..1'.›'..:'.'1' :.1'ufî'l ..w-;iguwamui i' .iiu i : war-.- i-.izi ; `5'.'35'>;I'v.' I Liv: :.;.-x;›'..~|.i~1~.~ ii isi-i niçulivßl'iiiil ; DIZI ID. No:295 Serial ID. NO:251 DIZI in. NO:99 um.' uavx1çcv-;L rs? :ci-vr::: :::defending n-;uzramvciun ama usa-'a ::u ;E'z' "JC'yJI'a'ILî-'I 'aL/.::1; IIIIVI n'i-;uiiviaimui : 32':: '31""v”l'CCP'LFS'-'SPCYJI'›'ILIC asi; ;::n'z: IIIFPI ::i ;QIPOEAFF 1.1.1: i ur i canin-ou› 1 :vs: asrin:: :49:22:: ne in'î expert n. i *i' ?al in 2;.: r-:c i- ar:: '. * L pm 1-. . 1 L :r: -i;_'.›; ;:;L'fl _ m inii ..vi-4.341 many. i LI i DIZI ID. No:. NO:99 :, ' iß'i'fYCFTJ'S'E'GF'R'RI'V'TLTE -SLIZ'JSS'IT'JGHFF'I ivf.“ :QXPGSÂP :14.13 9 :u inv- ::u array-suru L 5 : i-;-›::,i;i.rr; mmii Namazini-511.1› LIGHT cm: FRAME 3 m) FRAME 4 _ DlZl 1D. NO: 110 D1211D.NO: _r- SERIAL lD. NO: qui DIZl 1D. NO: but i !rl sleeps; +45 :-jvsps r-:›r›t.iz.eii~.uin Maçmcçso'iri- .Kisi CliP'i'i'E. ' WT-JLT-'W S :'31 :man: ?'PSPrr-snImemnunTxacûncçzo'nrr airiziirzvr. nuke-i.: -iß DIZIID.NO: iivn DIZIID.NO: '”78 DIZI ID.N():I(›Ü DIZI ID.NO:309 THS* :mycin: s'vwsr; ;ILülhJiALTIl'lRÜADEGAEi&'12 .urizirrz'cs Fuciitç--Lîum SEQ ID. NO: ii-:i SEQ ID.NO: liquid SEQ ID.N():I(›0 SequenceD.N0:309 ?Hand rabbit: C›'~_'P_[I_F rc m2. aka .nL 1' 1 :::kizima :5 'm- m Uzun's DECT-:1,746 Your ID NO: iia [)IZII[.NO: :im DIZIID.NO:160 DIZ_l ID. No:309 39113 iiriimn: ;~-rni~.r:-:;xLmimamniauntczowc ;un-:i: ?misra-i.?- in DIZI ID.NO: mi Array'ipNo; ;in SEQ ID4N01160 SEQ ID. No:309 nm: umran-_3 51-551". ;ILGIFMLII TUA'JADEHAL'LI'V. ALE'ILHPSUL ! DIZIIDÂNO: iiu DIZIID.NO: ,iis DIZI ID. No:160 SERIAL ID. No:309 SEQ ID. NO: 110 DIZIID.NO; ::in SEQ ID. No:160 DIZI ID No:309 but flour ::. ;product ;-:.:..1 expert.” ::Argi'ticizuunu ALt'JLlIl'L'i`L !Mugla-LI`- m Serial NO: 'im DIZIID.NO: '-.iui SerialD.N0;i60 SERIAL ID No:309 :] : match s-*Pnr- r-.scimiith II Iawmcew'if: Al. m ; fiber-'JKL legitimate-Lt'w l)lZ_11[)lNO: H., DIZllD.N0: -:,[internal DigiirgiNpgisp ArrayipipNozsos) ni: "T'ITF H: ?jppiprîzziLoibuLnTziicimceasi'ri' &LEICHP'F'SE nj-rçri:- '316 Table 15B: More than 94% total human identity with sequence identifiers for the full VL sequence VL amino acid sequences of germline-operated 27B3 clones. humanizing mutations are pale. Mutations with nrimer suffixes are in the same color as the buff.

. LIGHT FULL VL SEQUENCE ; I' '- 14.3 LIFE.' I'JCI'LJLI`~.~;:F'iiL'l 'JI'LICGUISGL'J'I LIIIIFE' WH' UUI i'CUfJ'kLLl 'i'llIllI M1:: 62'!' CUPL-Zg. IU.] ll'iAÄL'II ICMJADÜESEHE'CA LI' I :ICS'LJUEFD'JQI 0117140 GVF EGP::: ILGTEÄÂLTII :AQNJDE SDYFCWI 3 IIPCI'EE'GSGTQLT~'IG C'T'J'I'T OCPS LIVS?EST-'TLTCGUSGSI'TSTHFF TK!' ÜO'YPGWRLL 1 i l ITIISPMS 33'. &Fry .`32 3605 u.' 'sam-i.: lu.: IFRALTI î mumussz'u. L? 1 ;ini-;KJEPLLCI ULI'.'1A 234 350] çdi'i'i;Jüil'bPâi.i;['L:I'.'lLl'iuUZLLLi. ILuLFE'I'ni'IJUIiii'GIJAI'RLLI .::means .' 'ZT'Ü'TQEP with Jti Jfit :FIE'JPGETI .'.TCGLTJG :'"rswss'rn'ocincoapaunimum: G-'P :RTS-&S [W IRAN.? l 7 &OPIDCSC'YFCA L!' 2 :IIPZYEPGSGT OLT-'LG 2 3" 3662 GRI-"sTQEPJLF.'JPGST'-'TLICGLHJG CI'TZRIFP IWYQQIPGÇMRLLI'XIIIIITRHC (TFF I'RFI-SS I LG? TFT.? LT l T` i'iQFCDE :DYYCF LFI T. "PT VE'E'Ü'FGTQlTi'LG C-'F DEF ;GE I LC! lP'AÄL'II IMQFSÜESDYYCA LPI Z NF'L'i'EPGZIJYÇIL T-”LG GVF'DGFC '33 !DRTE'îPJL'IIIGAQPBUE SUYFCFNLF I 'J II? :-'x'EE'GSGTQLTVLG G`~'F pomegranate: 33 !MYFJJLTIIIÄHQPIDE :DYFCILLFI : II?C'L'EFOEGTQLT~`LG C-'F Kil-"F .I 33 I LG TFAAL'J'I IGÄLIADU!. &DYFCh LE`I S ICP'Â'- 'EF'GZIGTQLT'J W CUP DRTIT'R'. I 1;:: WiFi-.LT l TE& ÜWCP.! ?ICE L!' 1 .'iIIPIi't'EI'DI'î'iTC'l T~'LG 24-› 35k] Ç&'i'-.'JQEI'bFS'.';;F"L';T'i'I'LIClLU ;Gin ISLILFS'.\'i'UUIiC'GI.p`J 'Rl.LI illill RH: C-'P :PET-33 !Kalli/MIX T'JQÄDDE CDYICMPI S IIPCYEFGCIJIQLT'ILG 2'i_ SILL Ç'I'~'°i"IQEPSFSYSFGFI'-TLICGUIGGF-"IJINII'FIWXQQ'IFUCUJ'RLLITITIIITEKS Ci'F-IIRV'Z'TG I LG Wii-17 l TGÃOWÜE FGYYC& L!' I IIIIPSI'EFD'ZITOLT-”LG 14; Zilik: Oki".-IUî.I'IiP31r.-.F'L}Z.T'JTIJCLßUQÇLE' liiulFL' l'o x'UUIF'GUo'd'RLLI illIllI EH: 0-1" DGFC-GJ I mtamn :IRÜMDEAZ'SFCJLLPI î IIPZ'I'EP GDSTÇLT-'m Example 15: Temperature Stability of Germline Treated 27B3 mAbs Various germline-treated 27B3 mAb variants full-length human IgG1 molecules was expressed in HEK293 cells. After protein A purification The temperature stability of line-treated 27B3 mAb clones was evaluated. More To explain in detail, each mAh at various temperatures (2 or SCC intervals from each other) It was incubated for 1 hour in PBS buffer at a concentration of 100 tig/ml. Then a controlled The temperature of the mAbs was lowered in this manner (to 25°C within 2 hours, then overnight) Incubation was done at 4°C throughout) and centrifugation was used to remove aggregates. The amount of active mAb in the supernatant (as a percentage of total mAb concentration) with Biacore Results (shown in Table 17) most of the clones have wild type 27B3 It shows that it has a similar melting temperature (Tm). Unexpected appears to have.

Table 17: Temperature Stability of Germline Treated 27B3 mAbs mAb Clone Tm [OC] 64.7 64.6 35Gl 63.7 35G3 61.7 53A2 62.2 62.2 35G2 65.6 66.0 40Fl 63.5 53H1 64.1 Its stability was also studied using various techniques. Delbucco PBS with 0.02% Tween Representative amounts (1 m1) of test mAbs in a concentration of 100 tig/ml was prepared. Composed of buffer only (% one negative a control aliquot and mAb in Delbucco PBS containing 002% Tween. A representative amount of positive control was also prepared. Representative amounts of each mAb preparation At day 56 and 92, samples of 50 µl were taken for analysis from representative amounts. a sample It was stored at -20°C and used as a reference. .1 Analysis of Gel Filtration in Samples Samples taken at each time point were gelled using a Superdex200 10/300 GL column. added. A sample (125 µl) was taken for gel filtration analysis. Samples before analysis centrifuged to remove large aggregates. Monomeric peak percentage and monomeric peak area and also the volume of retention were measured. The results are shown in Tables 19, 20 and 21.

Table 19: Results of PBS Stability Study - Monomeric Peak %,si Table 20: Results of the PES Stability Study - Under the Monomeric Peak Curve Area (mAU*ml) Table 21: Results of PES Stability Study - Volume of Retention (ml) filtration analysis results are shown in Figure 137. A higher retention time (indicating that some aggregate is present) and a lower retention time (indicating some degradation product) two small peaks are seen. Results shows that most of the proteins are intact and do not form aggregates.

Analysis of Samples with BLACORE Samples from specific time points were allowed to bind to CD70 using Biacore. has been tested for its potency. 50 nl sample 200 u] PBS + 002% Tween,e was added and 5 al samples (1 mg/m1)+ as 1/400 diluted, then further diluted to 10 µl] + 90 nl of HVs-EP+ = 2.5 tig/ml.

Biacore analysis using a highly CD70 coated CMS chip is shown. Reference sample is 100% sample.

When the results are incubated at 37°C, the potency of the mAbs (antigen They show some loss of affinity for CD70. For 41D12 both tilt and maximum signal (R0) is marked (see Figures 16C and D). freeze-thaw stability was investigated. For this purpose, 0.7 ml of mAb31s (5 mg/ml) aliquot was frozen at -20°C for at least 6 hours and thawed at room temperature for 1 hour. This The cycle was repeated 9 times (in this way a total of 10 freeze-thaw cycles) and the samples were analyzed by gel filtration as above. Results for 10 cycles of mAb°s shows that they remain stable during freezing and thawing: they are degraded in gel filtration. products or aggregates could not be observed.

Table 22: Results of PES Gel Filtration Stability Study (left: 10 I/O cycles value after, right: value for reference sample) The samples were also tested for potency with Biacore as described above.

The results still have 971% of the potency of mAba after 10 freeze-thaw cycles. (for all of the three mAbs tested).

Table 23: Potency (%) of Biacoreida with 10 Freeze-Thaw Cycles 35G2 40Fl 41D12 Reference 100 100 100 Example 16: Germline Processed 27B3 mAb71erIII ADCC Effect Strength Several germline-treated variants of 27B3 as full-length human IgG1 molecules. It was expressed in HEK293 cells. Germline processing after protein A purification The ADCC potency of the 27B3 mAbs that were made was evaluated. described in Example 7 above was measured in .

Relative ICSOS of germline-treated 27B3 mAbs in Raji co-culture analysis (as described in Example 6) ADCC potency and CD70/CD27 blocking potency It is given in Table 18 by comparing it with parental 2783 in terms of < 1.0 a number indicates the enhanced IC50 of the germline-processed variant relative to parental 27B3. This Based on the data, the ADCC potency of the germline-processed variant 41D12° as well as the best Tm as well as the result that it retains its CD70/CD27 blocking power. can be removed.

Table 18: Germ in 786-0 Based ADCC Analysis by Comparison with Parental mAb Clone Relative IC50 of Line-Treated 27B3 Variants and in Raji-Based Bioanalysis Neutralization In the mAb Raji Common Culture Analysis, the Germ Line Process Was Made According to 27B3 ADCC ICSUS Related to IC50 mAbS by Clone 27B3 In the mAh Raji Common Culture Analysis, the Cherm Line Process was Made According to 27B3 ADCC 1C50 relative to IC50 mAb by Clone 27B39 Example 17: Unfucosylated CD70 mAh of ARGX-110 Expressing Cell Lines Making and Choosing The VH and VL amino acid sequences of the gerrn line-treated 27B3 clone 41D127. A binary gene vector encoding the za allotype was generated. 41D12j's heavy chain (SEQ ID.

No:344) and the nucleotide sequences encoding its light chain (SEQ ID No:345) are in the Table below. are also given.

Potelligent® CHOK] SV cells were stably grown with the dual gene vector by electroporation. was transfected. Six rounds of transfection were performed. of each electroporation in each round cells fattening medium was added to the CM633e and poured into forty 96-well plates at 50 ”L per well. covered. The selective agent L-methionine is added to each well the day after transfection. `150 µL of CM63 medium containing sulfoximine (MSX) was added to a final MSX of 50 µM. concentration was obtained. After approximately 3, 4 and 5 weeks of incubation, a cloning Using a mirror, the plates were scanned for the presence of colonies. Each defined colony, whether the colony originates from a single cell or from many cells examined separately using a microscope for evaluation.

Two hundred and forty-five colonies were identified using an Octet-based method and antibody production scanned for. Antibody concentrations ranged from 0 to 172 pg/mL. Scanned 245 214 of the cell lines were positive for antibody production. Cell lines by productivity graded and the first 70 cell lines were selected. Cultures of these 70 cell lines were in static culture. amplified and then suspension culture was done. Thirty years with acceptable growth cell line was selected and productivity screening with batch shake bottle culture analyzed in detail. Cultures were gassed on days 4, 6, 8, and 10, and collection on day 12. done. Antibody concentration in the collected supernatant sample Column containing Protein A determined by HPLC. Antibody in samples of 30 cell lines concentrations ranged from 109 to 877 mg/L. Intermittent shaking bottle evaluation The results were used to rank cell lines on the basis of productivity. first 20 cells line was selected for detailed evaluation.

Growth and growth of these 20 selected cell lines expressing ARGX-1 10 antibodies productivity data batch fed rinse bottle using CDACF media analyzed in cultures and the results are shown in Table 24. On the Protein A HPLC column The antibody concentrations at the time of collection determined were in the range of 857 to 3922 mg/L.

The F13 cell line was selected for grafting a disposable 10 L Cellbag bioreactor.

An antibody concentration of 4327 mg/L was obtained at the time of collection. This cell line is dashed An antibody concentration of 271 1 mg/L at the time of collection in the cultured shaker-fed and was second among all 6 cell lines (B1, D4, D5, F1, F13, and F18) examined. had the highest specific production rate (2.22 pg/cell/s). This cell line is accepted. exhibited acceptable growth characteristics.

Table 24: ARGX-Grown in CDACF Batch-Feed Shake Bottle Cultures l Growth and Productivity Data for 20 Selected Cell Lines Exhibiting 10 Antibodies .. Maximum Gathering Gathering Instant.. Specific Cell Live Cell Immediate IV Cm Immediate Antibody (2) Production(3I)-Trace Hatti Concentration 9.. Vitality Concentration (qp) (106/mL) (10 “ucre'S/L) (%) (mg/L) (pg/cell/s) Maximum To [but Collecting Instant.. Specific Cell Living Cell A p (1) Immediate Antibody Production Rate ninda IVC (2) (3) Line Concentration (109 cells s/L) Viability Concentration (qp) (106/mL) ' (%) (mg/L) (pg/cell/s) (1) Time integral of viable cell concentration at the time of collection. (2) Protein A is determined by HPLC. (3) The viable cell concentration of the antibody concentration according to the time integral. calculated by linear correlation analysis. Example 18: Alinity of CD70 mAbs 18.1 Affinity for CD70 Expressing Cancer Cell Lines Various cancer cell lines are present at saturated mAb concentrations (at least 2 µg/ml) of ARGX- described) was tested with. Analysis At 4°C (1 hour incubation of cells with mAbs) or at 37°C (15 minutes incubation of cells with mAbs). Anti-attachment hIgG1-Fc-FITC (AFOOÖ, Binding Site) or anti-hlgG1-Fc-PE antibody (eBioscience, 12- 4998) was detected using Fluorescence was measured using a flow cytometer. Results It is summarized in Table 25. In the third column of the table, the various tested variants of the ARGX-110 whether it binds to cell lines with low (+), medium (++) or high (+++) affinity. is shown.

In the right column, F ACS for ARGX-110 for the experiment at 37°C signal (MFI) is split into F ACS signal (MFI) for SGN70. These results are particularly that the high-affinity binding of the antibody can be more easily selected. When it comes to low copy number cell lines that can be expected, the ARGX-110° It shows that it binds to the cells with higher atintensity than SGN70.

Table 25: Binding of CD70 mAbs ARGX-1 10 and SGN703 to Cancer Cell Lines ARGX-II FACS Type Cell Line ARGX-110 signal/SGN70 F in ACS3 signal Large B-cell lymphoma SU-DHL-6 + 2.4 Non-Hodgkin lymphoma MHHPREBI +++ 1.2 Mantle cell lymphoma Chronic lymphocytic leukemia JVM-3 ++ In ARGX-l 10 FACS Type Cell Line ARGX-l 10 signals/SGN70 F in ACS signal Cutaneous T-cell lymphoma U266 +++ 0.8 multiple myeloma MM1.S++ KMSIZMB +++ 786-0 +++ 2.1 Renal cell carcinoma Caki-l ++ 1.9 A498++ 1.0 Astrocytoma U251+++ Lung carcinoma A549 + 3.8 WM1205-Lu+++ WM852+ Melanoma WM3248 +++ WM793+ WM1552C++ WM115+++ Glioblastoma U87M G++ 2-0 U343++ Ovarian carcinoma SK OV 3 Pancreatic carcinoma PANC ++ 2.9 In another experiment, CD70 mAbs, ARGX-10, SGN70 and Significant binding affinity of MDX14l lsin [see above] at various concentrations determined. Cells were incubated with a dilute batch of CD70-binding mAbder for 1 hour at 4°C. and binding was detected using anti-hIgG1-Fc-FITC or anti-hIgG-Fc-PE antibody. was done. Fluorescence was measured using a flow cytometer and the mean fluorescence was plotted.

The results are shown in Figure 15.

The results showed that the affinity of the three different mAbs for CD70 in cells was comparable. but MDXl4ll in some cell lines such as SU-DHL-6, A549 and MKN45 and the binding of the ARGX-101 was superior compared to the SGN70. SU-DHL-6, Bind to A549 and MKN45 other cell such as ECSOlsi, U266 and many others was higher for all mAb°s compared to ECSO in the probably that the mAb binds with only one arm (F ab), now avidity (binding strength) It shows that it does not provide and therefore lower attinities. Actually, The affinity of the ARGX-l 10 Fab relative to SGN70 and MDX14l when tested in Biacore much higher (see below). 18.2 Afmite for CD70 Expressing Patient Cells From chronic lymphocytic leukemia (CLL) patients (2 high-risk patients, 1 low-risk patient) harvested primary cells in RPMI 1640 + 10% PES with round bottom 96 wells plated at 250,000 cells/well and 5 tig/ml in RPMI + 10% PES. concentration of mAb91er added. After incubation at 37°C for up to 5 hours, cells washed once with PBS at ice cold. Goat Anti-Human marked with Alexa Fluor 647 incubated. The plate was washed twice with ice-cold PBS and left at room temperature for 15 minutes. Cells were fixed by adding 4% paraformaldehyde at temperature. Signals were measured by FACS.

The results are shown in Figure 167. Results belong to CLL patients of ARGX-110 It binds well to cells, while SGN70 gives almost no binding signal. shows. 18.3 Affinity of CD70 F abder for Recombinant CD70 in Biacore Recombinant human CD70 was fixed on a CMS Biacore chip. Fixing Biacore and using the NHS/EDC kit (Biacore AB) performed: 50 in 10 mM acetate buffer pH 5 after chip activation A µg/ml recombinant CD70 solution was prepared and 1 µl of this solution (50 ng) was injected. A surface density of approximately 1000 RU was obtained.

Fabs for CD70 mAb7s ARGX-1 10, MDX1411 and SGN70 via papain digestion was prepared. At a concentration of approximately [00-400 tig/ml, these F ab's are HEPES- buffered saline (0.1 M HEPES, 1.5 M NaCl, 30 mM EDTA, 0.5% v/v surfactant P20) was diluted 6-fold in They were injected (30µl) and flowed at a flow rate of 30µl/min. passed through their cells. A period of 10 minutes after the faba is connected to the CD70 The separation rate was monitored. Flow cell surfaces after dissociation 5 pl of 10 mM NaOH regenerated by injection. Sometimes resurfacing due to Fabalerin affinity multiple injections of NaOH were required. Dissociation rate analysis BlAevaluation It was done by applying to the software. First, the empty flow sensogram is obtained with the coated flow cell. removed from those. Then, use the Fit Kinetics app for a 10-minute walk. In the time interval, the separation rate was determined and the Kd value was calculated. Separation Rates Table It is summarized at 26.

Table 26: "Dissociation Rate" of Fabs for Connecting to Human CD70 Decomposition Rate [10'4 s"] Decomposition Rate [10'4 s"] 18.4 For Evaluation of Lysis in SU-DHL-6 Cells Binding With CD70 mAbs Loading Attempts SU-DHL-6 cells (a diffuse large B-cell lymphoma cell line) 50,000 It was pipetted into wells in the form of cells/wells, then 300,000 of healthy doners freshly isolated PBMC/well was loaded and different concentrations of nuÄb71er added. Samples were incubated for 2 days and checked for cell line depletion (anti- The best potency in terms of lysis was obtained with ARGX-l 10, which also gave 7% of the lysis.

The results are shown in Figure 17. Example 19: Efficacy of CD70 mAbs to Block CD27-CD70 The interaction between CD70 and CD27 promotes tumor cell survival, proliferation and/or may be associated with immunosuppression in the tumor microenvironment. Therefore, CD70 with ELISA The capacity of mAbs to block the interaction between CD70 and CD27 was examined.

In this analysis, a microtitration plate (Nunc Maxisorb) was first washed at 1250 fold in PBS. diluted (to obtain a final concentration of approximately 3.5 tig/ml) 100 111 with anti-FLAG M2 monoclonal antibody (Sigma Aldrich, F3165) overnight at 4°C. covered. The plate was washed once with PBS-Tween and 300 µl of PBS-1% for 2 hours at room temperature. was incubated with casein. The plate was washed again three times with PBS-Tween. PBS-0%.] casein was done. The plate was washed five times with PBS-Tween. The tested CD70 mAb was added to the plate; various concentrations by diluting the stock antibody solution in PBS-0.%] casein obtained. Immediately after this, 50 pl 1 tig/m1 ( recombinant human CD27 Fc chimera (R&D Systems 382-CD, MA = added and It was incubated for 1 hour at room temperature with shaking. The plate was washed five times with PBS-Tween. 13-0271) was added and the plate was incubated for another hour at room temperature with shaking. Number plate washed five times with PBS-Tween. 100 µl diluted 5000-fold in PBS-0.1% casein Strep-HRP (Jackson Immunoresearch 016-030-084) was added and the plate was shaken was incubated for another hour at temperature. The plate was washed five times with PBS-Tween. 100 u] TMB was added to the plate and OD was measured at 620 nm.

Using this analysis, the ARGX-110's blocking power is comparable to that of the SGN70 and MDX141 1 compared. As shown in Figure 18, the ARGX-l 10 is between CD70 and CD27. much more (approximately 100-fold) in blocking the interaction than the two comparison mAbs strong (IC50 ARGX-. Example 20: Binding Properties of CD70 mAbs .1 Cross-reactivity with Non-Human CD70 Determination of animal cross-reactivity of CD70 mAbs in vivo proof of concept which animal models can be used in studies and for toxicology studies. useful for assessing which species are the most suitable. A1 cheeks monkey CD70 is 94% identical to human CD70, while Cynomolgus monkey CD709i is human. It is 95% identical to CD70. An alignment of sequences of different species is in Figure 19 is shown. Mouse and mouse to highlight the differences compared to the human sequence Rat CD70 sequences are also included. U266iya (human multiple myeloma cell line), LCL8664, with a dilution series starting cells (rhesus monkey B-cell lymphoma cell line, ATCC-CRL-1805) and HSC- to F cells (cynomolgus monkey T cell line, fetal spleen-derived lymphocytes, Japanese Health Science Foundation) tested for attachment.

Detection was performed using goat anti-human IgGl FITC. Samples analyzed by FACS was done. The results are shown in Figure 20. of CD70 in both monkey cell lines. copy number is too low (low signals in FACS). However, the ARGX-l 10 SGN70 and MDX141 for cynomolgus and rhesus monkey CD70 It has a high affinity for CD70° when compared to its attenuation. .2 Blaking Power of CD70 mAbs its capacity to block the interaction is the blocking ELISA described in Example 197 above. tested using Analysis human. rhesus monkey and cynomolgus monkey Flag- Tailored to measure the blocking power of each antibody for TNC-CD70 (Wyzgol et al. ELISAida shows that IC50 values between different species can be directly compared. Exactly the same conditions were used for CD70 from the different species in question to ensure

The results are shown in Figure 21 and are applicable to humans, rhesus mines, and cynomolgus. This supports the results seen when cells from monkeys are used. ARGX-l 10 an unchanged potency and therefore the CD70°i of mines when compared to humans It has an unchanged affinity for the SGN70 and particularly the MDX1411 monkey CD70°i. It has a relatively low affinity for human CD707. IC50 values in Table 277 summarized.

Table 27: Blake Interaction of CD27-CD70 in ELISA for CD70 from Different Species 1C50 for Acquisition ._ Inhibition IC50 in ELISA [ng/ml] Cells . .3 Binding to Denatured CD70 Binding of CD70 mAbs to denatured recombinant CD70 was examined by ELISA.

Recombinant CD70 by heating at 95°C for 5 minutes, then immediately on ice for 5 minutes was denatured by cooling. A microtitration plate with or without denaturation coated with tig/ml CD70. The plate was blocked and the CD70 mAbs to be tested were 10 tig/ml was applied as After washing, mAbalinization using anti-human IgG-HRP binding detected. As a positive control for plating, use CD70 mAbs. Instead of using anti-F lag mouse, a mAb was administered and the detection was anti-mouse-HRP. was done with. TMB was used as substrate and OD was measured at 620 nm. This is ELISA It shows that while CD70 binds, other mAbs do not. Other mAb71er among others, some bind to denatured CD70 (fe 5F4, 9G2, 57B6). .4 Epitope Mapping Using F are-Human Chimeras By swapping human and mouse CD70 domains to map domain recognition of mAbs human-mouse CD70 ECD fusion proteins were generated. Generating standard recombinant It was done using DNA and PCR methodologies. Mouse and human chimeras in ELISA7 a Flag marker to capture and a TNC for trimerization of the protein (Flag-TNC-CD70) was cloned into a eukaryotic expression vector. Proteins are soluble in HEK293 cells expressed as proteins. The sequence of different chimeras is shown in Figure 23.

A Maxisorb (Nunc) microtitration plate 3.5 tig/ml mouse M2 anti-Flag mAb (Sigma Aldrich, F3165) and different chimeras were captured. CD70 mAbs to be tested a dilution series starting at a concentration of 10 tig/ml and making a 3-fold dilution Done. Binding after 2 hours Detection using anti-human-Fc-HRP at 16000 fold dilution was done. The dye was made with ABTS and the OD was measured at 405 nm. As a positive control human, with good affinity for both mouse CD70 and also chimeric human-mouse CD70 variants Binding CD27-Fe was applied. ECSO values for connecting are in Table 28 is summarized.

Table 28: ARGX-11 to Human-F are CD70 Chimeras shown in Figure 23° Connecting EC50 [ng/ml] ARGX-10 CD27 mouse NB 41 NB: no binding can connect but not to chimeras 2, 3 and 4 and mouse F'lag-TNC-CD70 shows. This indicates that the epitope for ARGX-110” is HIQVTLAICSS (SEQ ID.

NO:342) indicates that it is in the amino acid sequence. Example 21: Relating to Different Tumor Cell Lines and Primary Tumor Cells CD70 Internalization CD70 antibodies to 786-0 and A498 cell lines derived from renal carcinoma. binding leads to rapid (within 1 hour) internalization of the antibody-receptor complex. cultured in a well microtiter plate and incubated overnight at 37°C. was done. 2.5 tig/m1 mAb was added and incubated with cells at 37°C for 0-24 hours. given the code "INSIDE", which represents the amount of mAb that is internalized by PBS (representing the amount of mAb coded "DISARDA" and bound to the receptor outside the cell) was washed. After that, the cells were incubated with 4% paraformaldehyde for 30 minutes at room temperature. fixed, washed with PBS and 02 % Triton-X-IOO ("IN") or PBS for 5 min. ("DISARDA"). The cells were then washed twice and stored at room temperature for 10 minutes with 100 mM glycine followed by a 30 minute wash with PBS + 1% BSA. incubation was done. Finally, before analysis in the Li-Cor Odyssey infrared scanner cells goat anti-human FC (Jackson Immunoresearch 109-005-098) and anti-goat The results showed that the internalization for all mAbs was comparable and It shows that none of the mAbîs are completely intemalized. In the beginning, so Between 0 and 2 hours mAb internalization goes very fast but then a constant seems to be reached, even after 24 hours of incubation at 37°C. about 30% of the mAb remains outside the cell.

Internalization of CD70-bound mAbs can also be performed using other cell lines. examined. In these experiments, a test was performed using FACS analysis according to the protocol described below. As a time function, only the mAb signal outside the cell was measured. This alternative protocol It was determined to be a reliable reading when compared to the method described above. For suspension cells, cells were centrifuged, counted and 5 x 105 in pellet medium. cells/m1 were resuspended to a concentration and 100 uls of this suspension was 96 added to each well of a well V-bottom plate. Cells for adherent cells Seed into a 96-well plate and at 37°C and 5% CO until cells are completely adhered. enlarged. The cells provide linear cell growth for an additional 48 hours. spread in abundance. minutes, 5 minutes, 0 minutes (= without mAb) at 37°C, 5% CO incubated with cells. Following incubation, the plates were rinsed with cold FACS buffer. The internalization reaction was terminated by washing on ice. If you do at this stage cells were detached from the plate using cell separation solution (Sigma) and V-based 96 transferred to a well plate. The cells were then spun at 4°C. upper phase discarded by carefully inverting the plate. Cells were treated with 100 µl of the cell pellet by cold FACS. washed twice, carefully resuspending in buffer. after washing resuspended. The plate was incubated at 4°C for 30 minutes with shaking and the cells were washed once more with FACS buffer. Cells were resuspended in 100 µl F ACS buffer. suspended and fluorescence measured immediately. Intermediate/average Fluorescence intensity Some of the experiments were repeated twice or more. The results of the two experiments are quite was comparable. In some experiments, using U266, SU-DHL-6 and Raji cells, Whether the mAb concentration has an effect on the rate of internalization A concentration range (20 ng/ml - 5 µg/ml) was used to visualize. This kind of effect not observed (data not shown).

The results for the different cell lines tested are shown in Table 29. The results show that internalization is not a common event, in fact, it is an extremely rare event. proves that it happened. In Table 29, it is seen after 6 hours for different cell lines. the percentage of internalization is summarized. These results show that most of the ARGX-l 10 cells It shows that it remains attached to the outside of the cells, which makes the cells more adherent to ADCC, CDC and ADCP. tends to.

Table 29: % Internalization of CD70 mAb7s in Different Cell Lines After 6 Hours HH H Raw InteînîîgaîSEgîwsi Herâîtîî'gîîzlçm Burkitt's lymphoma Raji 2 2 Large B-cell SU-DHL-6 3 3 lymphoma Hodgkin lymphoma L428 non-Hodgkin 19 15 Hodgkin cell Mino 12 Le“fOma Granta 519 7 7 Chronic lymphocytic Mecl 3 leukemia JVM-2 8 Patient LR Cutaneous T cell HUT78 35 lymphoma HH 31 33 Multiple myeloma U266 38 MM1.S 35 Renal cell 786-0 58 carcinoma C aki-l 37 5 4 A498 66 Astrocytoma U251 52 52 Gastric carcinoma MKN-45 0 Lung carcinoma A549 3 Melanoma WM1205-Lu 45 44 THr H Heeer InteînîîîgaISîgîm H“Gîtîîîîîîzlçi" WM3248 34 Gliioblastino-Overnosal carcinoma 55134 U87MG carcinoma P ANC-89 20 25 Table 30: Nucleotide Sequences Encoding Selected CD70 Antibodies 41D12 Heavy chain WGÂGGYGCAOCTOGTGO AOICTOOG CGCCßiGGC'I' OCABGG MOGGCTCGAGAGTGGOT C1' CAGA'I'AGACTAGGACTAGGACTAGGACTAGGACTAGGACTAGGACTAGGACTAGGACTGACTGACTAGGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTGACTAGACTCAG TCCIGYTTCCTCCIAMCCCMAGACACTCTGAIGATIAGT CCGCTCCTAIAGAGMAACAATATCCMAGCCAAAGGTCMCXZTC GMCAACTACAAAACCACCCCTOCTGTTCTGGATTCTGACGGATC TTTCTTCCTTTATTCTAAACTCACCGTGGAIAAAAGCAGGTGGCAG IAACCATTATAOOCAGAAGTCTTTGTCCCICAGîOCAGGAAAGTG DEGISKEN BÖLGE LAMBDA SABIT BÖLGESI GCCTCAAATCTGGGTCTGTCACTTCCGATAACTTCCCCACTTGGT ACCAGCAGACACCAGGCCAGGCTCCCCGATTGCTTATCTACAA CACAAACACCCGTCACTCTGGCGTCCCCGACCGCTTCTCCGGAT CCATCCTGGGCAACAAAGCCGCCCTCACCATCACGGGGGCCCA GGCCGACGACGAGGCCGAATATTTCTGTGCTCTGTTCATAAGTA ATCCTAGTGTTGAGTTCGGCGGAGGGACCCAACTGACCGTCCTA TCAGAGGAACTGCAGGCCAATAAAGCCACACTCGTCTGCCTCAT CAGTGACTTCTACCCAGGAGCCGTGACCGTGGCCTGGAAAGCCG ACAGTAGCCCCGTGAAGGCCGGGGTGGAGACAACAACTCCTAGT AAACAGAGTAATAACAAATATGCCGCTAGTAGTTATCTCTCCCTCA CTCCCGAGCAGTGGAAGTCTCACAGAAGTTACTCTFGTCAGGTTA CTCACGAGGGTTCCACAGTGGAAAAGACTGTGGCCCCTACTGAA DEGISKEN BÖLGE LAMBDA SABIT BÖLGESI CCAGGAAAGGGGCTAGAGTGGGTCTCAGGTGATAATACCTACGA CAAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGG CTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTA GGTAGAGGCATTATGGGGGAGTACGGCAT GGACTACTGGGGCAA Hafif Zincir CAGACTGTGGTGACCCAGGAGCCGTCCCTGTCAGTGTCTCCAGG ACACCAGGCCAGGCTCCCCGATTGCTTATCTACAACACAAACAGC CCCAGTCGCTTCTCCGGATCCATCTCTGGGAACAAAGCCGCCCT CAGCCCGAGGACGAGGCCGACTATTACTGTGCTCTGTACATGGG GCTATGTTCGGCGGAGGGACCCATCTGACCGTCCTGGGTCA 59D10 Agir zincir GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTG TCCTGTGCAGCGTCCGAATTGTCCTTCAGTATTTCTGAGATGACC CCAGGAAAGGGGCTCGAGTGGGTCTCAGGTATTAGTGGTGTAAC AGTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGA TTGTATCTACAAATGAACAGCCTGATACCCGAGGACACGGCCGTA ACTAGTGGTACTTACTACTTCATCCCCGAGTATGAGTACTGGGGC Hafif Zincir CAGTCTGTGCTGACCCAGCCTCCCTCCGTGTCTGGGTCTCCAGG TCCTGTGCAGGAACCAGCAGTGATGTTGGGTATGGATACTATGTC TTCCCAGGAATGGCCCCCAAACTCCTGATATATGACGTCAATAAA GCTGATCGCTTCTCTGGCTCCAAGGCCGGCAACACTGCCTCCCT CAGTCTGAGGACGAGGCTGATTATTACTGTGCCTCATATAGAAGT TTCGGCGGAGGGACCCATCTGACCGTCCTGGGT DIZI LISTESI <1 10> arGEN-X BV <120> CD70 Antikorlari <130> NLW/P116378WOOO <160> 351 <170> PatentIn version 3.5 <210>1 <211> 30 <212>PRT <213> Lama glama <400> 1 Glu Lou Gln Val Vol Gl u Sir Gly Gly Gly Lou Vol Gln Pro Gly Gly l 5 10 15 25 30 <210> 2 <211> 30 <212> PRT <213> Lama glama <400> 2 Glu Vai Gln Lou Vil Glu Sir Gly Gly Gly Lou Val Gln? ro Gly 611 1 5 10 15 8.: Lou irq hu ask Cyi ni A1& 80: Gly Db. T: Ph. 80: 25 30 <210> 3 <211> 30 <212> PRT <213> Lama glama <400> 3 1 5 10 is 50: m &:9 bu 30: en AJ.. ani :0: Wear Db. rh: PM up 25 30 <210>4 <211> 30 <212>PRT <213> Lama glama <400> 4 Glu Val Gln Val Gin Glu Sir Gly Gly Gly Lou Val li. Pro Gly Gly 1 5 1 0 15 : m by. Lou :o: en AJ.. Mi 80: Wear Ph. rh: Ph. up 25 30 <210>5 <211> 30 <212>PRT <213> Llama glama <400> 5 Gln Vai Gln ! Au Val Glu Sir Gly Gly Mp Iiiiu !lal 6111 Pro Gly 611 l 5 10 15 80: hu end Im› ask Cy- Aii 11& S.: aiy Db. rh: vii 80: 25 30 <210> 6 <211> 30 <212> PRT <213> Lama glama <400> 6 Giu Vai Gin Ian Vai Gln Pro Gly Ala Giu ncu m Lyi Pro 61)' Ala 1 5 10 15 50: Vol Lyi Vii 30: Oya good A1; 80: Wear Ty: rh: Ph. m 25 30 <210>7 <211> 30 <212>PRT <213> Lama glama <400> 7 Sin ?al Gin Mu Gin Glu sir Wear Pro Wear mu vai good Pro 8.: Gin 1 5 10 15 th: Lou 8.!' Ian All Cyi I'br Year 8.: Gly Oly B.: II.. rh: 25 30 <210>8 <211> 30 <212>PRT <213> Lama glama <400> 8 Gln Val Gln ! au Val Glu sir Gly Wear Gly Inn m (Sin ?:0 Gly Gly 1 5 10 15 80: Lou Azg un 8.: Cyi Ala :mi End: Wear Db. ? hr Ph. Bu: 25 30 <210> 9 < 211> 30 <212> PRT <213> Lama glama <400> 9 Gln im Gln ni Vai Glu Sir Gly Gly Gly Lou m Gln Pro ciy, 50: m m Lou :o: en Mo Mi 80: Wear etc. rh: PM 80: <210> 10 <211>5 <212>PRT <213> Llama glama <400> 10 <210>11 <211>5 <212>PRT <213> Llama glama <400> 11 <210> 12 < 211>5 <212> PRT <213> Llama glama <400> 12 <210> 13 <211>5 <212>PRT <213> Llama glama <400> 13 <210> 14 <211>5 <212>PRT < 213> Llama glama <400> 14 <210> 15 <211>5 <212>PRT <213> Llama glama <400> l 5 <210> 16 <2ll>7 <212> PRT <213> Llama glama <400> 16 <210> 17 <211>5 <212> PRT <213> Lama glama <400> 17 <210> 18 <211>5 <212> PRT <213> Lama glama <400> 18 <210> 19 <211> 5 <212> PRT <213> Llama glama <400> 19 <210> 20 <21 1> 5 <212> PRT <213> Llama glama <400> 20 <210> 21 <211> 14 <212> PRT <213 > Lama glama <400> 21 Trp Val Arg Gln Ala Pro Arg Lys Gly Leu Glu Trp Val Ser <210> 22 <211> 14 <212> PRT <213> Lama glama <400> 22 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser <210> 23 <211> 14 <212> PRT <213> Lama glama <400> 23 Trp Val Arg Gln Ala Pro Gly Gln Wear Leu Glu Trp Met Gly <210> 24 <211> 14 <212> PRT <213> Lama glama <400> Arg Gln Pro Pro Gly Lys Gly Leu With 24 Trp Glu Trp Met Gly <210>25 <211> 14 <212> PRT <213> Lama glama <400> 25 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu With Trp Ser <210> 26 <211> 17 <212> PRT <213> Lama glama <400> 26 rh: ni 5. : rh:: Asp un si: &:9 th: ry: w: Al.. ! up 50: vii good 1 s 10 15 <210> 27 <211> 17 <212> PRT <213> Lama glama <400> 27 Asp 11. “n ”n Giu Wear Wear 'rhr rh:: :y: 1ryi: - m Asp Sir vii Lyi 1 5 1.0 15 <210> 28 <211> 17 <212> PRT <213> Lama glama <400> 28 hp Iio un nn Glu Gly up 'nu 'l'hr :yi: :yy : da np sir vii Lys 1 5 10 15 <210> 29 <211> 17 <212> PRT <213> Lama glama <400> 29 si: ni ryr Ilit ! yt up Su' si: th: ryr 'l'yr Al.. Asp Sor Vil well 1 5 10 15 <210> 30 <211> 17 <212> PRT <213> Lama glama <400> 30 Ala 11. :or 'rrp Su' 61:( 6.13( Ghz 'rhr Ph. tyr ni Glu p.: Int. well 1 5 10 15 <210>31 <211> 17 <212> PRT <213> Lama glama <400> 31 Giu ni rh: Sudden ?yt Wear 'ryr nn An; !yifyr ni Asp Sor vii good 1 5 10 15 <210> 32 <211> 17 <212> PRT <213> Lama glama <400> 32 ..:9 ! Type 1 Pro Glu Mp Gly Gly 'rhr Lyi Ty: AJ.: 6.111 Lyi PM 61:: 1 5 10 15 <210> 33 <211> 16 <212> PRT <213> Lama glama <400> 33 Ala ne 01 )' Se: Axq Gly Sor 'mx' 'ryr 'ryr Set ?to 891' Mu Lys 'rhr 1 5 10 is <210> 34 <211> 17 <212> PRT <213> Lama glama <400> 34 Asp ni nn Ask Wear Wear Wear Sir 'rhr good ryr m Asp 5.: 11.1 Lyi 1 5 10 15 <210> 35 <211> 17 <212> PRT <213> Lama glama <400> 35 Sir !1st Ty: Sir Alp Sir Sir!yt m!yt!yt Ala Asp Sir Va). good l 5 10 15 <210> 36 <211> 17 <212> PRT <213> Lama glama <400> 36 8.: ! 1. Tv: 8.:: Ilii 8.: Line ! yt th: ! yi- Yyr Ala Asp Sor Va). good l 5 1.0 15 <210> 37 <211> 17 <212> PRT <213> Lama glama <400> 37 Asp ni un un Giu Wear ! yt Giu rh: !yt ! yt Al.. Asp si: '1.1 by: 1 5 10 15 <210> 38 <211> 32 <212> PRT <213> Lama glama <400> 38 1 5 10 15 in nn The problem is well Asked Mp ! hr na vii Ty: ! yt Cyi Il. m 25 30 <210> 39 <211> 32 <212> PRT <213> Lama glama <400> 39 Am Ph. mi- n. Sir Ata Mp Ann Ala Lyi Ain rh: hu m hu Sin 1 5 10 15 25 30 <210> 40 <211> 32 <212> PRT <213> Lama glama <400> 40 Arq Ph. 'rr Il. Sir 1:; Mp nn Ala by. of 'l'hr ! Oh m! Au Sin 1 5 10 15 m up 801' ! ten by. ?:0 of the great hp. Lou Ty:: ! yt Cyi vii. m 25 30 <210>41 <211> 32 <212>PRT <213> Lama glama <400> 41 irq Ph. 'nu' Ilo sir rh: Mp Ann Ala Lyi Ain ! hr Vol 17!' Lou Sin 1 5 10 15 Hot un 30: Lou :good This is 'ali:up' nur ila vii ! yt 'ryi' ey. ili well 25 30 <210>42 <211> 32 <212> PRT <213> Lama glama <400> 42 m Ph. Tht 110 8.: fly un m Ala by. of TI::un ! yi- m 61:: 1 5 10 15 25 30 <210> 43 <211> 32 <212> PRT <213> Lama glama <400> 43 1 5 10 is 25 30 <210> 44 <211> 32 <212> PRT <213> Llama glama <400> 44 Arq Va). '1112 the rh: Ala Mp Ala Sir 111: 8.: rh: Ala ryr Val Glu 1 5 10 15 25 30 <210> 45 <211> 32 <212> PRT <213> Lama glama <400> 45 1 5 10 15 25 30 <210> 46 <211> 32 <212> PRT <213> Lama glama <400> 46 Arq PM Ala ! 1. 80: For Main Mp m Ala Lyi ! hr m ?yt im.: 61.0 1 5 10 15 HM: un 80: this Lyi Pro 611: hp 'rhr Ala vii ? yt ! yt 0,. Get. good 25 30 <210> 47 <211> 32 <212> PRT <213> Lama glama <400> 47 An) PM 'hu' :1. Sir i'm- Aip m Ala Lyi Ain ni: m 17:' hu 610 1 5 10 15 Mt un 8.: Lou :good ?:0 lap up 'Hu' Ala Vii ? eat it! yt q. Ala Gly 25 30 <210> 48 <211> 32 <212> PRT <213> Lama glama <400> 48 Arq Ph. 'mi' 110 Sir Arg Mp nn Ala by. of 'l'hr ! no! hr hu Sin 1 5 10 15 25 30 <210>49 <211>6 <212>PRT <213> Lama glama <400> 49 <210> 50 <211> 13 <212> PRT <213> Lama glama <400> 50 Asp Ala Gly Tyr Ser Asn His Val Pro With Phe Asp Ser <210> 51 <211> 19 <212> PRT <213> Lama glama <400> 51 Asp 11. &in irq 80: tv: Wear 80: 80: ? tp si: of Ph. Gly Pro II. 1 5 10 15 <210> 52 <211> 10 <212> PRT <213> Lama glama <400> 52 Wear Met Gly Leu Ala Giu Gly Leu Thr Asp <210> 53 <2ll> 10 <212> PRT <213> Lama glama <400> 53 Ser Leu Gly Leu Glu Tyr Gly Tyr Wear Asp <210> 54 <211>7 <212>PRT <213> Lama glama <400> 54 <210> 55 <211> 21 <212> PRT < 213> Llama glama <400> 55 vii Thr 617 610 Il. ru: Ty: Ann Sor Gly Sir ? yt ! yt Ty: ! hr Lou l 5 10 15 <210> 56 <211> 14 <212> PRT <213> Lama glama <400> 56 Glu Gly Gly Ser Gly Arg Tyr Trp Thr Asn Glu Tyr Asp Tyr <210> 57 <211> 10 < 212> PRT <213> Llama glama <400> 57 Ser Ser Asp Tyr Glu Gly Ser Phe Ala Ser <210> 58 <211> 10 <212> PRT <213> Llama glama <400> 58 Ser Ser Asp Tyr Giu Gly Leu Phe Vai Ser <210> 59 <211> 13 <212> PRT <213> Lama glama <400> 59 Asp Ala Gly Tyr Ser Asn His Val Gln With Phe Asp Ser <210> 60 <211> 11 <212> PRT < 213> Llama glama <400> 60 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser <210> 61 <211>11 <212>PRT <213> Llama glama <400> 61 Trp Gly Gln Gly Thr Gln Val Ile Val Ala Ser <210> 62 <211> 22 <212> PRT <213> Llama glama <400> 62 Gln ! tur Val vai ni: Gln Giu Pro :o: mu sir vu :n- ?:0 Gly 61, i 5 10 15 <210> 63 <211> 22 <212> PRT <213> Lama glama <400> 63 Gln &la Val Vol Thr Gln Gln Pro 8.: Lou Sir Val 3: Wear Pro 1 5 10 15 <210> 64 <211> 22 <212> PRT <213> Lama glama <400> 64 cm Ala Vai Vai 'nar uii Pro Pro 8.: Lou Sir iia 5.: Wear Pro Sir 1 5 10 is <210> 65 <211> 22 <212> PRT <213> Lama glama <400> 65 Ask Gln Ala hu ! hr Gln Pro Pro Sor ?lal Sor Gly 'l'hr Lou Giy Lyi 1 5 1.0 15 <210> 66 <211> 22 <212> PRT <213> Lama glama <400> 66 Gta 8.:: Ala ! nu ni: Gin Pro si: Ala Ve). si: Vol 80: m Gly Gin l 5 10 15 rh: mi &:9 II.. 'I'hr Oy. <210> 67 <211> 22 <212> PRT <213> Lama glama <400> 67 Gln 80:: Year m 'l'ht Gin Pro ?to 80: Lou 80: &li p.: ? ro Sir 80: l 5 10 ! 5 Sir vu Arq ! du 'Bu' Cyi <210> 68 <211> 22 <212> PRT <213> Lama glama <400> 68 Gln Ask: Ala Mu 'rhr Gln Pro Sir Ala mu Sir Val fb.: im Gly 610 1 S 10 ! 5 ml: n. ! good no 'mu Cy- <210> 69 <211> 22 <212> PRT <213> Lama glama <400> 69 cm sir Vai Vai 'l'hr Gin Pro Pro Ask Sir iia sir Pro Gly :or 1 5 10 is 802 Vil irq m '11:: Cyi <210> 70 <211> 22 <212> PRT <213> Lama glama <400> 70 su ua Va). !au 'l'hr 61.11 Pro Pro 80: ! su Sir &1: Sor Pro Gly :or 1 5 10 15 p.: vii fly flour 11:: Oy. <210> 71 <211>22 <212>PRT <213> Lama glama <400> 71 Gin na vii vii 'nu- Gln Glu Pro 30: ncu 'l'hr Val si: Pro Gly 61' 1 s 10 15 th : Vii nu' Mu 'nar Cy- <210> 72 <211> 22 <212> PRT <213> Lama glama <400> 72 Gin p.: Ala Ian 'rht Gln Pro 8.: th: Val Sir Vai p. :- hu 61)' 6111 1 5 10 15 <210> 73 <211> 22 <212> PRT <213> Lama glama <400> 73 Gin Pro Va). Mu rh: Gin Pro si: Ala Vol 8.:: Vol 80: hu Gly (Sin 1 5 10 15 th: Ala kg Il. 'nu' Oy. <210> 74 <211> 22 <212> PRT <213> Lama glama <400> 74 m Ph. Not !nu rh: Gln Pro 8.: Ala Val si: Vol 3: :Lou Gly Gln 1 5 10 15 th: Ala Arq IL. 'l'hr Cyi <210> 75 <211> 22 <212> PRT <213> Lama glama <400> 75 Gin Ala Val ! nu 'rhr Gln Pro si: 8.: Val Sir Vai Sir ! Au Glyi Gin 1 5 10 15 th: ni Lyi IJ.. ni: cy. <210> 76 <211> 22 <212> PRT <213> Lama glama <400> 76 cm Ala Vai !au 'i'hr Gin Pro si: Ask Vai Sir Val sir :au Gly Gin 1 5 10 15 <210> 77 <211> 14 <212>PRT <213> Lama glama <400> 77 Gly Leu Ser Ser Gly Ser Val Thr Thr Thr Asn Tyr Pro Gly <210> 78 <211> 14 <212> PRT <213> Lama glama <400> 78 Gly Leu Ser Ser Gly Ser Vai Thr Ser Ser His Tyr Pro Gly <210> 79 <211> 14 < 212> PRT <213> Lama glama <400> 79 Gly Leu Ser Ser Gly Ser Vai Thr Ser Ser Asn Tyr Pro Gly <210> 80 <211> 14 <212> PRT <213> Lama glama <400> 80 Gly Leu Thr Ser Gly Ser Vai Thr Ser Ser Asn Ty: Pro Asp <210> 81 <211> 14 <212> PRT <213> Lama glama <400> 81 Thr Leu Ile Ser Gly Asp Asn Ile Gly Gly Tyr Asp Iie Ser <210> 82 <211> 14 <212> PRT <213> Llama glama <400> 82 Ala Gly Thr Ser Ser Asp Val Gly Tyr Gly Asn Tyr Val Ser <210> 83 <211> ll <212> PRT <213> Llama glama < 400> 83 Gln Gly Gly Asn Ala Arg Phe Ser Ser Phe Ala <210> 84 <211> 14 <212> PRT <213> Lama glama <400> 84 Thr Leu Ser Ser Gly Asn Ser Val Gly Asn Tyr Asp Ile Ser < 210> 85 <211>11 <212>PRT <213> Llama glama <400> 85 Gln Gly Gly Arg Leu G ly Ser Ser Tyr Ala His <210> 86 <211> 14 <212> PRT <213> Lama glama <400> 86 Thr Leu Asn Ser Ala Asn Ser Vai Gly Ser Tyr Asp Ile Ser <210> 87 <211> 14 < 212> PRT <213> Lama glama <400> 87 Gly Leu Lys Ser Gly Ser Val Thr Ser Thr Asn Phe Pro Thr <210> 88 <211> 14 <212> PRT <213> Lama glama <400> 88 Gly Leu Thr Ser Gly Ser Val Thr Ser Asp Asn Phe Pro Val <210> 89 <211>11 <212> PRT <213> Lama glaina <400> 89 Arg Gly Asp Ser Leu Glu Arg Tyr Gly Thr Asn <210> 90 <211> 11 <212>PRT <213> Lama glama <400> 90 Arg Gly Asp Thr Leu Arg Asn Tyr His Ala Asn <210> 91 <211>7 <212>PRT <213> Lama glama <400> 91 <210> 92 <211>11 <212>PRT <213> Llama glama <400> 92 Gln Gly Gly Asn Leu Gly Leu Tyr Gly Ala Asn <210> 93 <211> ll <212> PRT <213> Llama glama <400> 93 Gln Gly Gly Asn Leu Trp Leu Tyr Gly Ala Asn <210> 94 <211> 14 <212> PRT <213> Lama glama <400> 94 Gly Leu Ser Ser Gly Ser Ala Thr Ser Gly Asn Tyr Pro Glu <210> 95 < 211> 14 <212> PRT <213> Lama glama <400> 95 Gly Leu Ser Ser Gly Ser Vai Thr Ser Ser Asn Tyr Pro Asp <210> 96 <211> 15 <212> PRT <213> Lama glama <400> 96 Trp Phe Gln Gln Thr Pro Gly Gln Ala Pro Arg Thr Leu Ile Tyr 1 5 10 15 <210> 97 <211> 15 <212>PRT <213> Llama glama <400> 97 Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu Leu Ile Phe 1 5 10 15 <210> 98 <211> 15 <212> PRT <213> Lama glama <400> 98 Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Val Leu With Tyr 1 5 10 15 <210> 99 <211> 15 <212> PRT <213> Lama glama <400> 99 Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15 <210> 100 <211> 15 <212>PRT <213> Llama glama <400> 100 Trp Tyr Gln Gln Lys Thr Wear Ser Pro Pro Arg Tyr Leu Leu Tyr 1 5 10 15 <210> 101 <211> 15 <212> PRT <213> Llama glama <400> 1 01 Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 102 <211 > 15 <212> PRT <213> Llama glama <400> 102 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Gln Val Ile Tyr 1 5 10 15 <210>103 <211> 15 <212> PRT <213> Llama glama <400> 103 Trp Tyr Gin Gln Lys Ala Wear Ser Pro Pro Arg Tyr Leu Leu Tyr 1 5 10 15 <210> 104 <211> 15 <212> PRT <213> Llama glama <400> 104 Trp Tyr Gln Gln Lys Pro Wear Gln Ala Pro Val Leu Val Ile Tyr 1 5 10 15 <210> 105 <211 > 15 <212> PRT <213> Llama glama <400> 105 Trp Tyr Arg Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 1 5 10 15 <210> 106 <211> 15 <212>PRT <213> Llama glama <400> 106 Trp Tyr Gln Gln Asn Pro Gly Arg Ala Pro Ile Leu Leu Ile Tyr 1 5 10 15 <210> 107 <211> 15 <212> PRT <213> Llama glama <400> 107 Trp Tyr Gln Gln Asn Pro Gly Arg Ala Pro Ile Leu Leu Phe Tyr 1 5 10 15 <210> 108 <211> 15 <212> PRT <213> Lama glama <400> 108 Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Leu Ile Tyr 1 5 10 15 <210> 109 <211>7 <212> PRT <213> Llama glama <400> 109 <210> 110 <211>7 <212> PRT <213> Llama glama <400> 1 10 <210> 111 <211>7 <212>PRT <213> Llama glama <400> 111 <210> 112 <211>11 <212>PRT <213> Llama glama <400> 1 12 Tyr Tyr Ser Asp Ser Tyr Lys His Gln Ser Ser <210> 113 <211> 7 <212> PRT <213> Llama glama <400> 1 13 <210> 114 <211>7 <212>PRT <213> Llama glama < 400> 1 14 <210> 115 <211>11 <212>PRT <213> Llama glama <400> 1 15 Tyr Tyr Ser Asp Ser Tyr Lys Asn Gin Gly Ser <210> 116 <211>7 <212> PRT < 213> Lama glama <400> 1 16 <210> 117 <211>11 <212> PRT <213> Lama glaina <400> 1 17 Tyr Tyr Ser Asp Ser Val Lys His Gln Gly Ser <210> 118 <211>11 <212>PRT <213> Llama glama <400> 1 1 8 Tyr Tyr Ser Asp Ser Leu Ser His Gln Gly Ser <210> 119 <211>7 <212> PRT <213> Lama glama <400> 1 19 <210 > 120 <211>7 <212> PRT <213> Llama glama <400> 120 <210> 121 <211>7 <212> PRT <213> Llama glama <400> 121 <210> 122 <211>7 <212 > PRT <213> Llama glama <400> 122 <210> 123 <211> 7 <212> PRT <213> Llama glama <400> 123 <210> 124 <211>7 <212> PRT <213> Llama glam < 400> 124 Vai Asn A511 A511 Arg Pro Ser <210> 125 <211>7 <212>PRT <213> Lama glama <400> 125 <210> 126 <211>7 <212>PRT <213> Lama glama <400 > 126 <210>127 <211> 7 <212> PRT <213> Llama glama <400> 127 <210> 128 <211>7 <212>PRT <213> Llama glama <400> 128 <210> 129 <211 > 32 <212> PRT <213> Lama glama <400> 129 Gly Vil. ! to Sir Mg Ph. 8.: My S.: 110 50: 01;( Main Member Ala Alti 1 5 10 15 tons ! hr ilc ! hr Gly ali Gln Pro Gln ! ip ol hi. Asp Ty: ! yi Cyo 25 30 <210> 130 < 211> 32 <212> PRT <213> Lama glama <400> 130 Gly Val Pro Sir &:9 ?yt Sir Gly 8.: II. 8.: Gly Ana by' Ala All Lou th: :1. th: aiy Al.. Gin ?:0 Sin np oiu Ala up ?yiryi: Oya 25 30 <210>131 <211> 32 <212> PRT <213> Lama glama <400> 131 Giy Val ?:0 Sir Arq Ph. Sir Wear 80: !1st Sir Wear Main Good Ala Al. 1 5 10 15 hu rh: !:10 th: Oly A1: Ola Pro Giu Asp en 111 hp ryr Vote !: 25 30 <210> 132 <211> 34 <212> PRT <213> Lama glama <400> 132 Wear vii Pro :0: Search Ph. Ask Wear 3.: by. Asp hic 8.: Al: Am Ala l 5 10 15 en m m Lou ne Ser ciy m Gin Ser Gln Mp Giu Ala Mp Ty: 25 30 <210>133 <211> 32 <212> PRT <213> Lama glama <400> 133 Gly Int Pro Asp Mg Ph. Water: Gly 80: by. 8.: Giy un ni: Ala 80: l 5 10 15 Lou ! lu- n. Sor aiy Lou Cin 80: 611: hp Ola iii up ! yi- good: Vote 25 30 <210> 134 <211> 32 <212> PRT < 213> Llama glama <400> 134 Wear 1. Pro Ala Mg Ph. Sir Gly 8.: So r Sir Wear It Ala Ala 'rhr 1 5 10 15 Lou ! hiria Sor Oiy Ala Oln ni Glu Asp Olu Ala up ! yi- ryr Oy. 25 30 <210> 135 <211> 34 <212> PRT <213> Lama glama <400> 135 Wear vii Pro :0: Arc Ph. Ask Wear 8.: by. &ip Pro 80: A1: Am Ala l 5 ID 15 Wear hu Mu ben 110 80: Wear Liu Gln Aii Glu Asp Giu Ala np 'a 25 30 <210> 136 <211> 32 <212> PRT <213> Lama glama < 400> 136 Wear ! Is lo Pro Glu tip. 5.: Gly :o: :o: si: Gly Mp m im n:: 1 s 10 15 i th: no 80: Giy ali @in AJ.. Ola Mp 010 iii Vol 'Good' fy: Can 25 30 < 210> 137 <211> 34 <212> PRT <213> Lama glama <400> 137 Gly vai ?:0 :o: Mo Ph. Sir: Gly 801' 80: &ip &in 8.: Ala Am Al. 1 5 10 15 Gly m m m n. sir Gly nu Gln !:0 Sin np Glu ua up Ty: 25 30 <210> 138 <211> 34 <212> PRT <213> Lama glama <400> 138 Wear vai Pro Ask Search Ph. Sor Gly 3.: rh: &ip Ala 80: Ala nin Al. 1 5 10 15 su m m hu ne 89: en Lan Gin Pro Glii Asp ciu mi Mp Ty: 25 30 <210> 139 <211> 32 <212> PRT <213> Lama glama <400> 139 Giy Vai Pro Sor Arg Ph . Sir Gly 8.: 11. :or Giu Ana Lyi Ala Al: 1 5 10 15 25 30 <210> 140 <211> 32 <212> PRT <213> Lama glama <400> 140 Gly Vil Pro Sir Arq Ph. Sir Gly 80: 21. th: Gly Ann by. Get it! li 1 5 10 15 Lou 'rbr n. rh: Dress up Cin Pro mu up ol Mi up 'ryr 'ryr Cyi 25 30 <210> 141 <211> 32 <212> PRT <213> Lama glama <400> 141 GI., ! 1. Pro Giu Arg Ph. Sor Gy 80: 8.: Sir Gy Ala nu: Aia Ala 1 5 10 is Lou ! hr ne 80: (ay Al. cin ni Glu Asp Ola Gly up ! yiryr Cyi 25 30 <210> 142 <211> 32 <212> PRT <213> Lama glama <400> 142 Gly ! 1. Pro Glu Arg Ph. Sir Gly 80: Arq Mu Gly Wear nu: Ala 'Bir 1 5 10 15 Lou th: vii p.: Oly ali six A1: 61:: Asp Ola Ala Mp Yy: 'I'yr Vote 25 30 <210 >143 <211> 32 <212>PRT <21 3> Lama glama <400> 143 Gly no Pro Gium ? ha 8.: Gly 80:' 11. p.: Wearing th: Ala '11): l 5 10 15 Lan rh: 1:10 80: aiy Ala (nn Va!. Giu Asp Giu Aii up ! yi-ry: Oy. 25 30 <210> 144 <211> 32 <212> PRT <213> Lama glama <400 > 144 Gly 110 Pro Glu Mg ?bu 8.:: Gly 80: :or 8.: Wearing Ala 'rhr l 5 10 15 hu ! hr n. 80: aiy Ala oin ni Giu Asp aiu Alan. ! yi- !yr Vote 25 30 <210> 145 <211> 32 <212> PRT <213> Llama glama <400> 145 617 ! 1. Pro Glu Mg ? what ? hr ! 10 80:' Lyi Sir Gly Gly Hu' Ala 'Hit 1 S 10 15 hu ! hr ti. Asp Oiy MJ Gin ua Giu Asp Ola 80: hp ! yt ! yt Vote. 25 30 <210> 146 <211> 32 <212> PRT <213> Llama glama <400> 146 Wear ! 1. Pro Glu Arc Ph. rb ! 10 80: by. Sir Gly Wear nu: Ala 'max 1 5 10 15 Lou rh: :1. kp aiy ni Oin ni Giu nn Giu sir up ryr ryr Oy. 25 30 <210> 147 <211> 32 <212> PRT <213> Lama glama <400> 147 Wear Vai Pro Wear Arg the sir Gly 80: 11. 8.: Gly Ana by. Ala Ala 1 5 10 15 Ldu th: Il. ! en Oly A1. Olu Pro Glu Asp Ola Ala up ryr 'l'yx' Oy. 25 30 <210> 148 <211>11 <212>PRT <213> Lama glama <400> 148 Ala Leu Glu Glu With Gly Ser Tyr Thr Tyr Met <210> 149 <211> ll <212>PRT <213> Lama glama <400> 149 Ala Leu Leu Asn With Asp Asp Gly Ser Thr Met <210> 150 <211> 10 <212> PRT <213> Lama glama <400> 150 Asn Leu His Leu Gly Ser Tyr Thr Pro Met <210> 151 <211> 12 <212> PRT <213> Llama glama <400> 15 1 Ala Leu Tyr Trp Gly Tyr Gly Thr Asn val Asp Val <210> 152 <211> 11 <212>PRT <213> Llama glama <400 > 152 Asn Leu Tyr Met Gly Ser Gly Gly Ser Lys Val <210> 153 <211> 12 <212> PRT <213> Lama glama <400> 153 Ser Ala Tyr Lys Ser Gly Ser Tyr Lys Ala Pro Val <210> 154 <211> 10 <212> PRT <213> Lama glama <400> 154 Ala Ser Tyr Thr Thr Asn Asn Lys Pro Val <210> 155 <211>9 <212>PRT <213> Lama glama <400> 155 Gin Ser Tyr Giu Ser Glama Asn Tyr Vai <210> 156 <211>11 <212>PRT <213> Lama glama <400> 156 Ser Val Ser Asn Ser Gly Thr Tyr Lys Pro Val <210> 157 <211> 10 <212> PRT <213> Llama glama <400> 157 Gln Ser Gly Ser Ser Asn Thr Asn Val Met <210> 158 <211 >9 <212> PRT <213> Lama glaina <400> l 5 8 Ser Ala Tyr Lys Ser Gly Ser His Vai <210> 159 <211>9 <212> PRT <213> Lama glama <400> 159 Ser Ala Tyr Asn Arg Gly Ser His Val <210> 160 <211> 10 <212> PRT <213> Lama glama <400> 160 Ala Leu Phe Ile Ser Asn Pro Ser Val Glu <210> 161 <211> 10 <212>PRT < 213> Llama glama <400> 161 Ala Leu Tyr Leu Giu Asn Phe Ala Asn Glu <210> 162 <211> 10 <212> PRT <213> Lama glama <400> 162 Gln Ser Ala Asp Ser Ser Gly Asn Ala Val < 210> 163 <211> ll <212>PRT <213> Llama glama <400> 163 Gln Ser Ser Asp Ser Ser Gly Tyr Arg Val Val <210> 164 <211> 10 <212> PRT <213> Llama glama <400 > 164 Gln Ser Gly Ser Ser Ser Thr With Pro Val <210> 165 <211> 10 <212> PRT <213> Lama glama <400> 165 Gln Ser Ala Asp Tyr Ser Giy Asn Ser Val <210> 166 <211> 10 <212>PRT <213> Lama glama <400> 166 Gln Ser Ala Asp Tyr Ser Giy Asn Ser Val <210> 167 <211> 10 <212> PRT <213> Lama glama <400> 167 Gln Ser Ser Asp Tyr Pro Gly Asn Ser Val <210> 168 <211> 12 <212> PRT <213> Lama glama <400> 168 Leu Leu Tyr Met Gly Gly Ser Asp Phe Asn Phe Vai <210> 169 <211> ll <212>PRT <213> Lama glama <400> 169 Phe Gly Gly Gly Thr His Leu Thr Vai Leu Gly <210> 170 <211>11 <212>PRT <213> Lama glama <400> 170 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly <210> 171 <211> ll <212>PRT <213> Lama glama <400> 171 Phe Gly Gly Gly Thr Thr Leu Thr Val Leu Gly <210>172 <211>11 <212> PRT <213> Lama glaina <400> 172 Phe Gly Gly Gly Ser Lys Leu Thr Val Leu Gly <210> 173 <211>11 <212>PRT <213> Lama glama <400> 173 Phe Gly Gly Gly Thr His Leu Thr Val Leu Ser <210> 174 <211>11 <212> PRT <213> Lama glama <400> 174 Phe Gly Gly Gly Thr Gin Leu Thr Val Leu Ser <210> 175 <211>11 <212>PRT <213> Lama glama <400> 175 Phe Gly Gly Gly Thr Arg Leu Thr vai Leu Gly <210> 176 <211>11 <212>PRT <213> Lama glama <400> 176 Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly <210> 177 <211> 115 <212> PRT <213> Lama glama <400> 177 Glu 14.0 (Sin Lou Oin Itir. 11 am; 01, <210> 178 <211> 122 <212>PRT 3.: en : me <213> Lama glama <400> 178 6130 611 M9 ini 61.!! 8.: &ip 11. va). m Asp <210> 179 <211> 122 <212>PRT <213> Lama glama <400> 179 80: Gly the Giunpfh: M. 308 80: Trp Glu vii Gln iau ni Glu Sor Gly Gly Gly mu Val Gln Pro Gly Gly : m m Lou 30: cn u. mi :o:aiy etc. r: mi. :o: vu.Ty: tyr Hut Atti 14: Biy Arg <210> 180 <211> 128 <212> PRT <213> Lama glama <400> 180 8.: I 1.

GLi !lot <210>181 <211> 119 110 Years. #3: Wear 611 Ask A1 Mp 8.: Sir #3: "If #8: Gly 1 of them:: 101.! <212> PRT <213> Llama glam <400> 181 8.: Take; 11th. <210> 182 <211> 119 <212> PRT <213> Llama glam <400> 182 9.1 Ty: 80: Lou Ana 80: Bin 110 <210> 183 <211> 116 <212> PRT <213> Llama glam <400> 183 611 Aip 611 Gly Giu vii Gln Lou vii. Gln Pro 80: vu Lyi vu So: Cyi good six months vu rh:: Ph. m Vdßluuusir Sirmlng 110 Sor Ara Arq Arq Mp Ph. <210>184 <211> 131 <212>PRT <213> Llama glam <400> 184 80: 01)› Ty: !hz 617 'rhr good !yt Giu Mp 1):: Al.

Gin Vol Gln Lou @in Giu Ser Giy !to Gly Lou vii what is this 80: @in tin-Mus.: rrpuicaiy <210>185 <211>123 <212> PRT <213> Llama glam <400> 185 Gln Va). Sin 8.: type I!..

The 81st & lu& 1'11» Ala well Glu Gly Wear Sir Gly Aug ?yt ?tp Tht Ain 610 !yt Asp Ty: <210> 186 <211> 119 <212> PRT <213> Llama glam <400> 186 <210>187 <211>119 <212>PRT : Vote. .: medicine <213> Llama glam <400> 187 : my what 611 Gly 80: 50: <210> 188 <211> 122 <212> PRT 589 ?yt <213> Llama glam <400> 1 8 8 Ty: 'l'hr 'ryi- <210>189 <211> 112 <212>PRT <213> Llama glam <400> 189 <210> 190 <211> 112 <212> PRT <213> Llama glam <400> 1 90 <210> 191 <211> 111 <212>PRT <213> Llama glam <400> 191 Gin rh: Vai Vai rh: Gln Glu Pro so: Lou si: Vol 8.: Wear Pro Gly 40 45 m II. Ty: Hello fb: Mo un m it:: 3.: Oly vu Pro 80: M0 Ty: 65 70 75 00 Bin ?to Olu Asp Oiu li; lap ty: Ty: Cyi !in Lan Rii Lou aly :o: 85 90 95 Ty: !hr !:0 Hit Ph. Wear Oly: Lyi Lou !hr Vii Ldu Bir 100 105 110 <210> 192 <211> 113 <212> PRT <213> Llama glam <400> 192 1 5 10 15 m vii 'mx nu rh: Cyi Gly uu -nu: ask Gly Sir vu nu.- :o:30: Ain Tv: ?:0 lap ?29 ?yt s Gin fb: !to Gin Ala Pro Arc Lou co work Lou Ile Ty: non ?hr aan Sor Atv “to Set Gly Vol Pro 50: Ata Ph. 50 55 60 Sor ciy Ask !10 Ask Ask Aon good A1; 110 Lou The !1. Tour Gly A1. 65 70 75 50 Gin ?:0 Sin Asp Glu ala Asp !yt !yt Cys nin &en 2:: type Gly Ty: 05 90 95 dir th: !in VII &ip vii Ph. Gly Wear Gly Tbr good ncu !hr Vil Lou <210> 193 <211> 112 <212> PRT <213> Llama glam <400> 1 93 80: Wear 30: <210> 194 <211> 119 <212> PRT <213> Llama glam <400> 194 aiii ini %1 Ve). rh: im Sir Vairq 92° 80:' Main 80: ua ryr <210>195 <211> 111 <212>PRT #3: Oiy <213> Llama glam <400> 195 mi ben 'i'hr with Se: Cyo <210> 196 11th:.! <211> 107 <212> PRT <213> Llama glam <400> 196 A1. frp 'ryr Gin Gin by. hp great Ala Aip 'ryr ?yi- <210> 197 <211> 118 <212>PRT <213> Llama glam <400> 197 Gin sir Va). Mu'l'hr 6111 m 1.00 Ty: ?yi- 'ryr Ask 11.. Pro M9 Pro 811 Gins 80: PM 613! V.! Lou Lou ne :o: 617 hu Gin Ala Glu Asp Giu ala Asp !yt Ty: Oya 85 90 95 100 105 110 <210> 198 <211> 108 <212> PRT <213> Llama glam <400> 198 l 5 10 15 25 30 40 45 :5 60 65 70 15 80 Hp 0111 A1: Va). Ty: 'l'yr Cyi Gin 8.: Wear Boz Bu Mu nu' un Va!. 85 90 95 iht Ph. Gly Gly Gly th: Bii !Au ni: VI). Lou: 100 105 <210> 199 <211> 116 <212> PRT <213> Llama glam <400> 199 Gln: Val Va). rh: Gln Pro Pro 8.: Lou Sir Ala Sir ?:0 Gly Sir 1 5 10 15 25 30 <210> 200 <211> 116 <212> PRT <213> Llama glam <400> 200 Gln Ala Yil. 14`:th sir na !yi- <210>201 <211> 111 .one." <212> PRT <213> Llama glam <400> 201 6111 Ala Va). ask ciy 80:' <210>202 <211> 111 <212>PRT Va). ?hr (Sin 110 301' CN <213> Llama glam <400> 202 6111 110 Vai Va). rhr Gin Vol frp 'l'yr 119 t'hr Gly Phe Ala Asn Glu Phe Gly Gly Gly Thr Arg Leu Thr Val Leu Gly <210>203 <211> 108 <212> PRT <213> Llama glam <400> 203 <210> 204 <211> 109 <212> PRT <213> Llama glam <400> 204 6111 8.: Ala !nu rh: m Ala An; 1:.. rh: 11. ?:0 1115 Pro 8.:: Val Gly' Speed 511 610 65 70 75 80 Mp Ola Mi Asp Ty: Ty:: Cyi Ola 30: :0: type 50:30:' &y !ye- m 85 90 95 Va). Va). Ph. Gly Oiy Oly ! is good this 'l'hr Val Lou Oiy 100 105 <210> 205 <211> 104 <212> PRT <213> Llama glam <400> 205 1s 10 15 25 30 en by: Wear Pro Gin Mi Pro 931 i vii !10 !ye ne Ma na un 40 jobs m what :o: Oiy no ?:0 extreme PM :0: month :0: no :o: m, M0 55 60 m M.: '111: i rh: ne 80: &y ua Gin vii Grass up Giu M.. asp 65 70 15 80 nm Ty: Cyi Possible: my Bu 80:' p.: 'rhr II. ?:0 Wow!. PM Oly 01.3 85 90 95 Gly Bu: Lyi hu th: V1). Wear Lou <210> 206 <211>104 <212> PRT <213> Llama glam <400> 206 Ann Ph. Note !nu rh: Gln Pro 8.: Ala Val Sir Vai Sir !Au Gly Gin l 5 10 15 th: Ala W 11. Thr Oy. 012: Gly Oly 'l'yr Ty: !hz an 711:› !yt Gln 25 30 6111 by. ?:0 th: ia 'rhx 611( Bu' Lyi <210> 207 <211> 108 <212> PRT Ask Gly 80:' <213> Llama glam <400> 207 6111 iii Va). un ri-p month: 013( hp flour van. gly <210> 208 <211> 108 <212> PRT A::: Pro Lou Gly 'Hu' good <213> Llama glam <400> 208 ÄIJ'I ll'l M0 80: Gly Mu Glu Ala AJ.. number 80: 6131 Moment: 5th: 80:' 019 <210> 209 <211> 108 <212> PRT <213> Llama glam <400> 209 80: algae <210>210 <211> 113 1111' <212> PRT <213> Llama glam <400> 210 80: Gly :o: <210> 211 <211> 113 <212>PRT 110 80: <213> Llama glam <400> 21 1 #3: &mg 65 70 75 00 01:› ?:0 0.111 up ulu ni Mp ?yt l'yr Cyi ni Lou ?yt Int Wear 30: Oiy 8.: ”n un Vai Va.! Ph. My Wear My 'mu' Oiu Lou mr vil. I 100 105 110 <210> 212 <211>122 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 212 1 5 10 15 25 30 good- )Iit un Trp vii An; mn Ala Pro Wear well Oiy Ian Glu frp vu 00 45 8.:- tip ni Ma Ana Glu Gly Gly 'rhr 'rhr ?vr ?yt Ala Asp Sor Va). 50 55 60 by. Gly Arg PM 'l'hr rlo 60: Search up kon 50: by: Ma !'hr !ou 'I'hr 65 70 75 80 hu Oin !hi nin 80: un good !to Giu Aip rh: ni vii ryr !yt Cyi 85 90 95 Va). m Asp nlu Gly ryr 8.: m mi Va). Pro : 1. Ph. Aip Sor 'ri-p 100 105 110 115 120 <210> 213 <211> 122 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 213 Glu Va). 6111 hu 801' Lou Au; Lou Ty: !lot m 'rrp 01;: this: aiy 'rhr <210> 214 <211> 122 <212> PRT <213> Artificial <220> Mi 30: Wear ?ho AJ.- Pto Ily good Gly 'rhr 'rhr Ty: m ”9 Sudden A1. un n Va). Pro <223> Germ Line Processed Antibody Array <400> 214 611 611 Ask 'ti-p 8.: &ip Iic <210> 215 <2ll> 122 <212> PRT <2 13> Artificial <220> 80: Gly <223> Germ Line Processed Antibody Array <400> 215 50: Type 80!' this 8.: &ip 6130 @In <210> 216 <211> 122 <212> PRT <213> Artificial <220> Val Ari; 8.: Lou 611 617 80:' Gly 86!' :er <223> Germ Line Processed Antibody Array <400> 2 1 6 611 Gly 611: vii Gln Lou Vil. Gln si: Gly Gly Gly me 80: Im› m Lou So: Cyi Al.. A1. 80: 01)› the 8.: &ip 11. lan this Glu Gly Gly 'rhr 'rhr 'ryr 65 10 75 muted Sirmiyil'ro Giunpfh: na m Asp Ala Gly ryr 80: m mi vii Pro 100 105 Oiy mi› 017 rh: Lou vii m vii :o: :o: 115 120 <210> 217 <211> 122 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 217 Glu Vai Gin !au Val Glu sir Gly 613! 61°/ I 89: m Atv hu 80: Oya Ala Mi 80: Wear Ph. ryr Int nn ?tp vii M'q Cin Ala Pro Wear Ly- 8.2 Mp Il. un &in GLu Oiy my ni: ?hr Ty: good Gly im ?hi rh: 11. Water: An; “9 Mi:si: 611 Gly 80:' 'Hp 80:' V::. 65 70 75 00 85 90 95 AJ.: m Mp Ah Oiy ryr 80: m lu.. Vii no no Ph. up 30: Type 100 105 110 613( (:10 Wear 'I'hr m Vol ?m- 901 Ask sir 115 120 <210> 218 <211>122 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 218 Glu vii Gin !nu vii Glu Sir Gly 617 611( Lou vu Gln Pro Gly Gly 1 5 10 jobs Ask !Au &to hu Sor Cyi Mi 80: 80:* su Ph. 'Itir Ph. Ask vii Ty: 25 30 13:: Min: un 'ti-p Year An; Gln Al: !:0 Wear it well aiy Ian Glu frp V.! 40 45 80: “p 11. nn un 610 Gly Wear 'rhr 'i'hr Ty: !yt Alo Aq› Sor vii 50 55 60 by. Gly AN PM 'I'hr No Sir Are np han :0: by. Ma ni: that's 'Good' 65 70 75 80 Ask Lou mn In Lou Arg mi 310 Aip ?hz ni vu !yi- !yr Cyi 85 90 95 Va). m Asp Ala Gly ?yt 5.: m il Va). Pro !1. Ph. 1.09 8.: 'ri-p 100 105 110 115 120 <210> 219 <211> 122 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 219 Glu Va). Gln huh 801' Lou Au; Lou Ty: !lot m 'rrp 01;: this: aiy 'rhr <210> 220 <211> 122 <212> PRT <213> Artificial <220> : Cyi AJ..

Year. Arc Oin Wear 'Pvr Sir 611 Gly !mu : wear the of Va). Pro 80: :o: <223> Germ Line Processed Antibody Array <400> 220 611 611 Ask 'ti-p 80: what hp <210> 221 <211> 122 <212> PRT <2 13> Artificial <220> <223> Germ Line Processed Antibody Array <400> 221 80!' this 8.: &ip 6130 @In <210> 222 <211> 122 <212> PRT <213> Artificial <220> Val Ari; 8.: Lou 611 617 80:' Gly 86!' :er <223> Germ Line Processed Antibody Array <400> 222 611 Gly 611: vii Gln Lou Vil. Gln si: Gly Gly Gly me 80: vu m Lou So: Cyi Al.. A1. 80: 01)› the 8.: &ip 11. lan this Glu Gly Gly 'rhr 'rhr 'ryr 65 10 75 muted Sirmiyil'ro Giunpfh: na m Asp Ala Gly ryr 80: m mi vii Pro 100 105 Oiy mi› 017 rh: Lou vii m vii Jun :0: <210> 223 <211> 122 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 223 Glu Vai Gin !au Val Glu sir Gly 613! 61°/ I 89: m Atv hu 80: Oya Ala Mi 80: Wear Ph. ryr Int nn ?tp vii M'q Cin Ala Pro Wear Ly- 8.2 Mp Il. un &in GLu Oiy my ni: ?hr Ty: good Gly im ?hi rh: 11. Water: An; “9 Mi:si: 611 Gly 80:' 'Hp 80:' V::. 65 70 75 00 85 90 95 AJ.: m Mp Ah Oiy ryr 80: m lu.. Vii no no Ph. up 30: Type 100 105 110 613( (:10 Wear 'I'hr m Vol ?m- 901 Ask sir 115 120 <210> 224 <211> 122 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 224 1 5 10 jobs Ask !Au &to hu Sor Cyi Ma Mi Sor Wear Ph. 'Itir Ph. Ask vii Ty: 25 30 13:: Min: un 'ti-p Year An; Gln Al: !:0 Wear it well aiy Ian Glu frp V.! 40 45 80: “p 11. nn un 610 Gly Wear 'rhr 'i'hr Ty: !yt Alo Aq› Sor vii 50 55 60 by. Gly AN PM 'I'hr No Sir Are np han :0: by. Ma ni: that's 'Good' 65 70 75 80 Ask Lou mn In Lou Arg mi 310 Aip ?hz ni vu !yi- !yr Cyi 85 90 95 Ala m Asp Ala Gly ?yt 5.: m il Va). Pro !1. Ph. 1.09 8.: 'ri-p 100 105 110 115 120 <210> 225 <211> 122 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 225 Glu Va). Gln huh 801' Lou Au; Lou Ty: !lot m 'rrp 01;: this: aiy 'rhr <210> 226 <211> 122 <212> PRT <213> Artificial <220> Mi 30: Wear ?ho AJ.- Pto Ily good Gly 'rhr 'rhr Ty: m ”9 Sudden A1. un n Va). Pro <223> Germ Line Processed Antibody Array <400> 226 611 611 Ask 'ti-p 8.: &ip Iic <210> 227 <2ll> 122 <212> PRT <2 13> Artificial <220> 80: Gly <223> Germ Line Processed Antibody Array <400> 227 50: Type 80!' this 8.: &ip 6130 @In <210> 228 <211> 122 <212> PRT <213> Artificial <220> Val Ari; 8.: Lou 611 617 80:' Gly 86!' :er <223> Germ Line Processed Antibody Array <400> 228 611 Gly 80: Im› m Lou So: Cyi Al.. A1. 80: 01)› the 8.: &ip 11. lan this Glu Gly Gly 'rhr 'rhr 'ryr 65 10 75 mmmunsirmmniaiunprh: na m Asp Ala Gly ryr 80: m mi vii Pro 100 105 115 iso <210> 229 <211> 122 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 229 Glu Vai Gin !au !ml II.: 8.1' Gly 613! 61°/ I 89: m Atv hu 80: Oya Ala Mi 80: Wear Ph. ryr Int nn ?tp vii M'q Cin Ala Pro Wear Ly- 8.2 Mp Il. un &in GLu Oiy my ni: ?hr Ty: good Gly im ?hi rh: 11. Water: An; “9 Mi:si: 611 Gly 80:' 'Hp Val 6111 Pro Gly 617 good flour 'mr m 'ryr 65 70 75 00 85 90 95 AJ.: m Mp Ah Oiy ryr 80: m lu.. Vii no no Ph. up 30: Type 100 105 110 613( (:10 Wear 'I'hr 'I'm- Vol ?m- 901 Ask sir 115 120 <210>230 <211> 111 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 230 Gin nu' Va). Va). rh: Gln Glu Pro 8.: rh. Ask Val 8.: Pro Gly Gly 1 5 10 15 m vii 'mr Lou rh: Cyo Wear !good :o: Wear Se: Vei nu- Se: m 25 30 M0 PM Pro TM' frp ?yt Ol!! 61:! 'I'm- Wear Pro @In A!. Pro hcg Lou nn no Ty: nn fl:: im: m m mi 80: aiy voi im› up Mg PM 55 60 65 70 75 80 Bin Ala np Aip Oiu Su' Mp ?yt PM Cyi ua Lou an. 11.. 8.:”n 85 90 95 100 105 110 <210>231 <211> 111 <212>PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 231 m Va] '17:: me <210> 232 <211> 111 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 232 611 611 80: Main 8.: viii <210>233 <211> lll <212> PRT <213> Artificial <220> 11:: flour Water: am 80: up 91:. (month 011( 141.! Sat Lan 'l'hr il Ask Wear <223> Germ Line Processed Antibody Array <400> 233 80: Wear ?to Gly vai this: Sir 'rhr Pro Asp Ari; ?ho 61:: This.' Va). Va). r: m Year n: Lou rh:: Ma PM Pro 'l'hr frp 110 i <210> 234 <211> 111 <212> PRT <213> Artificial <220> M0 80: m 013/ flour Lyo 8.: type !yt <223> Germ Line Processed Antibody Array <400> 234 Gin Ala vii Va). 'Mar m 9.1 '111: Lou Thr 110 !yt Mu ?hr p.: Wear 8..: :li's Oya 81 :( neu A1. Glu ?yt 3.: ?:0 at 110' That Gly Ph. i.. Boots vii : Wear Lou 8.2 Pro 611 ni 920 M4: 11. &:9 31 :( vil. I'm Gly <210> 235 <211> 111 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 235 Gln l'hr Va). vii rh: Gin 6111 Pro 8.: rh. sir Vol 8.: Pro Gly Gly 1 5 10 15 m:- vii 'rhr Iiou 'l'hr Cyi Gly !ou Lyi Sor Giy Sor vol 'I'hir Sor Mp 25 30 Main PM Pro 'I'hr 'l'rp ?good- Gln Gln 'I'hr Pro Wear Gin A1. Pro M'g Lou 00 45 Lou no Ty: Main fb: MA 30: m im :o: Oiy Vol Pro Asp Mg PM 50 55 60 65 10 75 00 of Ala up Asp Giu ua mu ryr 'ryr Cyi ni un Ph. 1:10 80: flour 05 90 95 100 105 1.10 <210> 236 <21 l> lll <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 236 Gln nu: Va). <210>237 <211> 111 <212>PRT <213> Artificial <220> rh:: Cyi Gly 91:. Gly Gly 80: Gly Ser : Gly V“ <223> Germ Line Processed Antibody Array <400> 237 Gln Ala Va).

Va). ? hr Gln Glu Pro Sir Lou 'rhr Vai hu 'I'hr Oy. Gly Lou by: unrbrunmirqnii Asp Giu p.: Asp good: 'l'yr <210> 238 <211> 111 <212> PRT <213> Artificial <220> <223> Germ Line Assayed Antibody Array <400> 23 8 Gln! hr Val vii rhr Gin Giu ?to a.: Lou rh: Vol 8.: Pro Gly Gly 1 5 10 15 th: vii ? hr Lou fhr Wear Cyi Vol ? hr Ask Wear Ask vol ! hr Ask pill 25 30 han ? ho Pro Thr frp Ty: Gln Gln ? ni Pro Wear Gln A1. Pro Irc Lou Lou Il. Ty: Main fb: han th: Inc 8:: :o: Oiy Vol Pro Aip Atq etc. 50 5: 60 : Wear So: Iio Lan Wear &sn ;ya aia ala ben th: With rh: Wear: 65 10 75 00 Thousand Pro Great Alpine Gin 8.: lap Ty: Tyr Cyl Ala Lan Ph. II. 8.! Main 85 90 95 100 105 110 <210> 239 <2l l> 111 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 239 Gln Ala Val Val Thr Gln Glu Pro 8.: Ph. Sir Vol 8.: Pro 611 Sir 1 5 10 [5 rh: VII ! hr neu? hr Wear Cys Lou rh: Ser Gly Se: vai rh: Ser Thr 25 30 00 45 I 110 ! yt un 'l'ht Ata rh:: m ini 80( Wear Vol 50 55 60 S.: Wear 8.:: 11. Inn Giy un Lyi Ala Ala Liii :rh: 65 70 75 en ni Asp Asp Giu nin en îy : Ph. Oyu Ala hu 100 105 <210> 240 <211> 111 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 240 6111 Bu Vai Va). rbr Gln 611.: Pro Sor Ph. Sir m: Vii 'mr Lou 'l'hr Oy. 613 Lou by: Sor Giy 80: Mn PM Pro 'I'hr frp fyr Gin Gin 'Nu' Pro Giy Gin Mu Il.. Ty: nin 11:: Lu› 80: Am mi :o: Oiy mu 55 60 : My Se: ne Lou uy un Lys ni mi Lou ni: 65 70 75 Cin Ali Mp Alp Giu Sir Mp 'l'yr Ph. Cyi Ala bu Pro 8.: vii Glu Ph- Oiy Oiy Giy 'rhr Iln mn ! hz 100 105 <210> 241 <211> 111 <212> PRT <213> Artificial <220> 8.: ”n 611 611 80: nn <223> Germline Processed Antibody Array <400> 241 6121 &li Va). <210>242 <211>111 <212>PRT <213> Artificial <220> i'br Gln Glu i'm- Cyo Gly frp ? yi- Gin 1.00 Oiy un <223> Germline Processed Antibody Array <400> 242 what 9.1 ? hr Water 61:; Set 0.1!! Fm 310 Pro Ser val Glu Phe Gly Gly Gly Gly Thr Gln Leu Thr Val Leu Gly <210> 243 <211> 111 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 243 Gln Hu' Va). Va). i'br mi- Vii 'This' Lou 'i'm- un PM Pro 'I'br frp 80: Wear 80: ne Lou Pro 8.1' V1). Glu? this is <210> 244 <2l l> 111 <212> PRT <213> Artificial <220> <223> Genn Line Processed Antibody Array <400> 244 <210> 245 <211>111 <212>PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 245 8.: Wear 5.: 80: Asp 8.: ..o 611 611 8.: Asu <210> 246 <211> 111 <212> PRT <213> Artificial <220> <223> Germline Analyzed Antibody Array <400> 246 Gln Ala Vol vii rh: Gin Giu ?to a.: Ph. sir Vol 8.: Pro Gly Gly 1 5 10 15 th: vii ? hr Lou fhr Cyi Gly ! au Lyi Sor Wear Ask vol ! hr Ask pill 25 30 han Pho Pro ? hr frp Ty: Gln Gln ! hr Pro Wear Gin A1. Pro Arc Lou to 45 Lou Il. Ty: Main fb: han ra: Ang HL: :o: Oiy Vol Pro 3: Atq etc. 50 5: 60 : Wear So: Iio Lan Wear &sn &ya aia ala ben th: Ile rh: Wear: 65 10 75 00 Thousand li: Asp Alp Cin 8.: lap Ty: Tyr Cyl Ala Lan Ph. II. 8.! Main 85 90 95 100 105 110 <210> 247 <21 l> lll <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 247 Gin ! hr vii vii rh: Gln Glu Pro 8.: Ph. si: Vol 8.: Pro Gly Gly 1 s 10 is 25 30 un Ph. Pro 'rhr rrp I'yr mn Gin 'rhi' Pro Oly Oin Ala Pro Arq Lou 40 45 un 11: ! rhrnnthtizqnii BirGlyVnJ of the year. ?to ScruqPhi 50 55 60 Sar Gly Sex 113 &en Gly !um by; Ala Ala ben rh: With This' Wear A1. 65 70 75 IIO nin &li up Aip Oiu Sir Mp 'I'yr 'ryz Cyi Ala Inn PM› 110 80: M0 85 90 95 Pro Ser val Glu Phe Gly Gly Gly Thr Gln Leu Thr val Leu Gly 100 105 110 <210> 248 <211> 5 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 248 <210> 249 <211> 17 <212> PRT <213> Artificial <220> <223 > Germline Processed Antibody Array <400> 249 lap 11. Ana Ann Glu Gly Gly ala rh: !yt ty: &1.3 heat› Sor Va). by: 1 5 10 15 <210> 250 <211> 14 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 250 Gly Leu Lys Ser Gly Ser Val Thr Ser Asp Asn Phe Pro Thr <210> 251 <211> 14 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 251 Gly Leu Thr Ser Gly Ser Val Thr Ser Thr Asn Phe Pro Thr <210 > 252 <211> 14 <212> PRT <213> Artificial <220> <223> Genn Line Processed Antibody Array <400> 252 Gly Leu Thr Ser Gly Ser Val Thr Ser Asp Asn Phe Pro Thr <210> 253 <21 l> 14 <212> PRT <213> Artificial <220> <223> Germline-Treated Antibody Array <400> 253 Gly Val Thr Ser Gly Ser Val Thr Ser Asp Asn Phe Pro Thr <210> 254 <211> 30 < 212> PRT <213> Lama glama <400> 254 61:: im Gin nu vii Giu su- Gly Gly Gly mu ni 61:: ?to Wear 61; 1 5 10 is Sat Lou &:9 m 80:: Oy. ni Mi 80: Gly etc. 50: Ph. 80: 25 30 <210> 255 <211> 30 <212> PRT <213> Lama glama <400> 255 Glu Val 6111 !nu Vol Glu Sir Gly Gly Gly Liu Val Gln ? to Gly One 1 5 10 ! 5 Sir Lou &:9 ! au 80: Vote. ni in 3:: Glu Lou Sor Ph. This is: 25 30 <210> 256 <211>5 <212> PRT <213> Llama glama <400> 256 <210> 257 <211>5 <212> PRT <213> Llama glama <400> 257 <210>258 <211> 17 <212>PRT <213> Llama glama <400> 25 8 Wear np nn rh:: ! yt np Wear Wear ! hr km 'speed Gin Asp p.: Vii good 1 5 10 15 <210> 259 <211> 19 <212> PRT <213> Lama glama <400> 259 Wear Il. :o: Gly Yil. m Gly Gly 80: S.: 'I'bt Sir ty: Ala Asp Set 1 5 10 15 <210> 260 <211> 32 <212> PRT <213> Lama glama <400> 260 l 5 10 15 25 30 < 210> 261 <211> 32 <212> PRT <213> Lama glama <400> 261 Arq Ph. ? hr ! 1. Sir Atv Alp by Ann Alp. ain ! hr Ian :y: iiu 610 1 5 10 15 Ain 80: Lou ti. Be a Pro Aip ? hr Ala vii ? yt ! yt Cyi Ala rh: 25 30 <210> 262 <211> 14 <212> PRT <213> Lama glama <400> 262 Asp Thr Gly Arg Gly With Met Gly Glu Tyr Gly Met Asp Tyr <210> 263 <211> 13 <212> PRT <213> Lama glama <400> 263 Thr Ser Gly Thr Tyr Tyr Phe Ile Pro Glu Tyr Glu Tyr <210>264 <211>11 <212>PRT <213> Lama glama <400> 264 Trp Wear Lys Gly Thr Leu Vai Thr Val Ser Ser <210> 265 <211> 22 <212> PRT <213> Lama glama <400> 265 Gln ! hr val Vai? hr Gln Gln ?to :o: i 8.: Vol si: Pro Gly Gly l 5 10 15 rh: Val ! hr ! du 'Bir Cyi <210> 266 <211> 22 <212> PRT <213> Lama glama <400> 266 Gln Sir Val nin ? hr Gln Pro Pro Sor 911 Sir Gly Sor Pro Gly Lyt 1 5 10 15 <210> 267 <211> 14 <212> PRT <213> Lama glama <400> 267 Giy Leu Lys Ser Gly Ser Vai Thr Ser Ser Asn Tyr Pro Ala <210> 268 <211> 14 <212> PRT <213> Lama glama <400> 268 Ala Gly Thr Ser Ser Asp Val Gly Tyr Gly Tyr Tyr Val Ser <210>269 <211> 15 <212>PRT <213 > Llama glama <400> 269 Trp Tyr Gln Gln Phe Pro Gly Met Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 270 <211>7 <212> PRT <213> Lama glama <400> 270 <210> 271 <211> 12 <212> PRT <213> Lama glama <400> 271 Ala Leu Tyr Met Gly Ser Gly Ser Ala Asn Ala Met <210> 272 <211> 32 <212> PRT <21 3> Lama glama <400 > 272 Gly ! lo Ala Asp Arq ? ha 5.: Gly 80: by' Ala Gly Main th: Ala &or 1 5 10 15 Lou th: 1:10 8.: aly un 01:: 80:: @lu Mp ol Ala up ! yt ! 7: Vote. 25 30 <210> 273 <211> 10 <212> PRT <213> Lama glama <400> 273 Ala Ser Tyr Arg Ser Ser Ala Asn Ala Val <210>274 <211> 123 <212>PRT <213> Artificial < 220> <223> Germline Processed Antibody Array <400> 274 619 hp Atti 619' m Phe <210> 275 <211> 124 <212> PRT <213> Artificial <220> 3.: Embroidery 11:: Ala 019 ? yt <223> Germline-Treated Antibody Sequence <400> 275 p.: Ilii Ty: <210>276 <211>113 <212>PRT <213> Artificial <220> <223> Germline-Treated Antibody Sequence <400 > 276 6111 nu: Val Vol rh: Gln Glu m Vol 'm2 Lou rh: Cyi 01.3 Bin Pro 811: Asp ulu Ala Mp <210> 277 <211> 111 <212> PRT <213> Artificial <220> Ala hu ? yt ! Int Oly 50: <223> Germline Processed Antibody Array <400> 27 7 I 110 no:- Mp vu nn well 6111 Ph. Pro Sir Mp V1). Wear ? yt Wear Gly With! no 920 Lyi ben su 110 Alo Mp Arc PM Gin 80: 610 Asp Gin Ala Mp 'ryi' 'ryt Cyi ala si: ? yt m 8.: 80: 85 90 95 ml “n A1. Va). Ph. Gly Bly Gly 'rhr ini Lou rh: vii hu aiy 100 105 110 <210> 278 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Treated Antibody Array <400> 278 1 5 10 1.5 im: un Sor Lou good Pro ali› np nu- Ala Ian 'ryz ! yt Cyi vu. m 25 30 <210> 279 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 279 Arq Ph. 'mi' 110 Water: tip Mp m Ala Lyi Ain TI:: un ! yt Inn Sin 1 5 1.0 15 note Ann 80:' Lou good Pro 01:› hp This' &1. Lou Ty: ! yt Cyi vu m 25 30 <210> 280 <21 1> 32 <212> PRT <2l3> Artificial <220> <223> Germline Processed Antibody Array <400> 280 Arq PM 'This' ! 1. 80: Search Mp m 80: by. &in Sir m m m 61:: 1 5 10 is 25 30 <210> 281 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 281 1 5 10 15 im: Un take the lead ni› Asp nu- ua vii 'ryz ! yt Cyi vu. m 25 30 <210> 282 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 282 Arq Ph. 'mi' 110 Water: tip Mp m Ala Lyi &in 3-: !aci nubiu Sin 1 5 10 15 note Ann 80:' Lou kg iii 01:› hp Bu' &1. vii Ty: ! yt Cyi vu m 25 30 <210> 283 <21 1> 32 <212> PRT <2l3> Artificial <220> <223> Germline Processed Antibody Array <400> 283 Arq PM 'This' ! 1. 80: Search Mp m Ala by. &in TI:: m ! yt m 61:: l 5 10 15 25 30 <210> 284 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 284 1 5 10 1.5 im: flour Ask Lou good Pro ali› np nu- Ala vii 'ryz ! yt Cyi ni m 25 30 <210> 285 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 285 Arq Ph. 'mi' 110 Water: tip Mp m 80:' Lyi &in 3-: ! pain ! yt Inn Sin 1 5 1.0 15 note Ann 80:' Lou kg iii 01:› hp Bu' &1. vii Ty: ! yt Cyi ni m 25 30 <210> 286 <21 1> 32 <212> PRT <2l3> Artificial <220> <223> Germline Processed Antibody Array <400> 286 1 5 10 is 25 30 <210> 287 < 211> 11 <212> PRT <2l3> Artificial <220> <223> Germline Processed Antibody Array <400> 287 Trp Gly Gln Gly Thr Gln vai Ile Val Ser Ser <210> 288 <211> ll <212> PRT <213> Artificial <220> <223> Genn Line Treated Antibody Array <400> 288 Trp Gly Gln Gly Thr Gln Val Thr Val Ala Ser <210> 289 <21 l> ll <212> PRT <213> Artificial <220 > <223> Germline Treated Antibody Sequence <400> 289 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser <210> 290 <211> 11 <212> PRT <213> Artificial <220> <223> Germline Treatment Fabricated Antibody Array <400> 290 Trp Gly Gln Gly Thr Thr Val With Val Ser Ser <210> 291 <211> 11 <212> PRT <213> Artificial <220> <223> Ship Line Processed Antibody Array <400> 291 Trp Gly Gln Gly Thr Thr Val Thr Val Ala Ser <210> 292 <211> ll <212> PRT <213> Artificial <220> <223> Germline-Treated Antibody Array <400> 292 Trp Gly G ln Gly Thr Gln Val Thr Val Ser Ser <210> 293 <211> I I <212> PRT <213> Artificial <220> <223> Germline-Treated Antibody Array <400> 293 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser <210> 294 <21 1> 22 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 294 Gln nu: Val vii rh: Gln (Sin ?:0 :o : ini sir Val si: Pro Wear sir i s 10 15 rh: Vii th: !au 'This' Cy- <210> 295 <21 1> 22 <212> PRT <213> Artificial <220> <223> Germ Line Process Made Antibody Array <400> 295 Gin :ua vii vii ni: Gln Glu Pro 8.: mi.. :o: Val sir Pro Gly Wear 1 s 10 15 rh:: ViJ. ! hr Mu 'Speed Oy. <210> 296 <21 1> 22 <212> PRT <21 3> Artificial <220> <223> Germline Processed Antibody Array <400> 296 Gln ! hr val Va). 'l'ht Gin Giu ?to 80: Lou TI:: Vol Sir ?:0 Sir Wear 5 10 ! 5 11:: V“ m Ian :rh: C3!. <210> 297 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 297 Wear Vai Pro Asp &9 rh. Sir Gly S.: 11. ! Wear nn by. Ala Ala 1 5 10 15 Lou th: n. 'No- Wear Aii Oiii A1; up up 010 :o: Mp ry: DM Oya 25 30 <210> 298 <211> 32 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 298 Gly Val Pro si: Arc Ph. sir Gly 80:: ! li Lou Oiy m good Ala Al. 1 5 1.0 15 Louth: n. n:: Gy M.. Oin iui up up 010 50: Mp ry: DM Oya 25 30 <210> 299 <21 1> 32 <212> PRT <2l3> Artificial <220> <223> Germ Line Processed Antibody Series <400> 299 Gly Vol Pro Aq› Mg ? this is 8.:: Gly :o: Il. Lou Dress m by: Ala Ala l 5 10 15 1.00 th: Il. Arc Oly A1. Ola Mi Au: MP 010 A1.. Ola !yt Ty: Oy. 25 30 <210> 300 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 300 Wear Vai Pro Asp &9 rh. Sir Gly S.: 11. ! Wear nn by. Ala Ala 1 5 10 15 Lou rh:: n. m: 01)› ni Cin Pro Glu Asp Giu 80: hp ryr Ph. Vote. 25 30 <210> 301 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 301 Gly Val Pro Asp Arc Ph. sir Gly 80:: ! li Lou Oiy m good Ala Al. 1 5 1.0 15 Lanth: ::1. rh: an 11.. 01:: ?:0 Gin np ol 50: hp ! yi- ryi: Oy. 25 30 <210> 302 <21 1> 32 <212> PRT <2l3> Artificial <220> <223> Germline Processed Antibody Array <400> 302 Gly Vol Pro 8.: Mg ? this is 8.:: Gly :o: Il. Wear Lou by: Ala Ala l 5 10 15 hu ! hr : 1. rh: aiy Ala oin ni up Asp aiu Ala Giu ! yi- Mn Oy. 25 30 <210> 303 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 303 Wear Vai Pro Asp &9 rh. Sir Gly S.: 11. ! Wear nn by. Ala Ala 1 5 10 15 Lou th: n. 'No- Wear Aii Oiii A1; up up 010 Mi formation fy: DM Oya 25 30 <210> 304 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 304 Gly Val Pro Asp Arc Ph. sir Gly 80:: ! li Lou Oiy m good Ala Al. 1 5 1.0 15 Louth: n. n:: Giy M.. Oin iui Mp up 010 im Mp ry: DM Oya 25 30 <210> 305 <21 1> 32 <212> PRT <2l3> Artificial <220> <223> Germ Line Processed Antibody Series < 400> 305 Gly vii Pro 8.: Arg ? this 8.: Gly 80: 11. I Wear Asu good Ala Alo l 5 10 15 Lou th: IJ.. Hu' Oly Mi Ola Mi Mp Mp Grass 50:' hp ?yt Ty: Oy. 25 30 <210> 306 <211> 17 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <220> <221> VARIOUS_FEATURES <222> (1)..( 1) <223 > X is any amino acid, preferably D, T, S or E, <220> <221> VARIOUS_FEATURE <222> (2)..( 2) <223> X is any amino acid, preferably I <220 > <221> VARIOUS_FEATURE <222> (3)..( 3) <223> X is any amino acid, preferably N, S, T or Y <220> <221> VARIOUS FEATURE <222> (4).. (4) <223> X is any amino acid, preferably N, M, S or T, <220> <221> VARIOUS_FEATURE <222> (5)..( 5) <223> X is any amino acid, preferably E is D, Y or H <220> <221> VARIOUS_FEATURE <222> (6)..( 6) <223> X is any amino acid, preferably G, D, S or N <220> < 221> VARIOUS_FEATURE <222> (7)..( 7) <223> X is any amino acid, preferably G, Y, S, D or M5 <220> <221> VARIOUS_FEATURE <222> (8)..( 8 ) <223> X is any amino acid, preferably T, E, S, N, Y or R7 <220> <221> VARIOUS_FEATURE <222> (9)..( 9) <223> X is any r is amino acid, preferably T, A or R <220> <221> VARIETY FEATURE <222> (17)..(17 ) <223> X is any amino acid, preferably G or S <400> 306 xnmmmmnnmuaniiyrwriiamsirwiiyi 1 5 10 15 <210> 307 <211> 5 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <220> <221> VARIOUS_FEATURE <222> (1)..( 1) <223> X is any amino acid, preferably V, G or A <400> 307 <210> 308 <211> 5 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Sequence < 220> <221> VARIOUS FEATURE <222> (1)..( 1) <223> X is any amino acid, preferably D, T, S, N or G <220> <221> VARIOUS_FEATURE <222> (2 )..( 2) <223> X is any amino acid, preferably Y, S or P* <400> 308 <210> 309 <211> 11 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <400> 309 Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly <210> 310 <211>7 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Array <220 > <221> VARIOUS_FEATURES <222> (1)..( 1) <2 23> X is any amino acid, preferably N or S <220> <221> VARIOUS_FEATURE <222> (3)..( 3) <223> X is any amino acid, preferably N, S or A < 220> <221> VARIOUS_FEATURE <222> (4)..( 4) <223> X is any amino acid, preferably S, N or T <400> 310 <210> 311 <211> ll <212>PRT <213 >Artificial <220> <223> Germline Processed Antibody Sequence <220> <221> VARIOUS_FEATURE <222> (6)..( 6) <223> X is any amino acid, preferably Y, V or L" < 220> <221> VARIOUS_FEATURE <222> (7)..( 7) <223> X is any amino acid, preferably K or S <220> <221> VARIOUS_FEATURE <222> (8)..( 8) <223> X is any amino acid, preferably H or N <220> <221> VARIOUS_FEATURE <222> (10)..( 10) <223> X is any amino acid, preferably G or S <400> 31 1 Tyr Tyr Ser Asp Ser Xaa Xaa Xaa Gln Xaa Ser <210> 312 <211>7 <212>PRT <213> Artificial < 220> <223> Germline Processed Antibody Sequence <220> <221> VARIOUS_FEATURE <222> (1)..( 1) <223> X is any amino acid, preferably V, 1 or Y <220> < 221> VARIOUS_FEATURE <222> (3)..( 3) <223> X is any amino acid, preferably N or T <400> 312 <210>313 <211>7 <212> PRT <213> Artificial < 220> <223> Germline Processed Antibody Sequence <220> <221> VARIOUS_FEATURE <222> (4)..( 4) <223> X is any amino acid, preferably Y <220> <221> VARIOUS_FEATURE <222> (5)..( 5) <223> X is any amino acid, preferably R or M <400> 313 <210> 314 <211>7 <212> PRT <213> Artificial <220> <223> Germ Line Processed Antibody Sequence <220> <221> VARIOUS_FEATURE <222> (1)..( 1) <223> X is any amino acid, preferably D or G <220> <221> VARIOUS_FEATURE <222> (4 )..( 4) <223> X is any amino acid, preferably S or P <400> 3 14 <210> 315 <211> 14 <212>PRT <213> Artificial <220> <223> Germline Processed Antibody Array <220> <221> VARIOUS_FEATURE <222> (3)..( 3 ) <223> X is any amino acid, preferably S or T <220> <221> VARIOUS_FEATURE <222> (7)..( 7) <223> X is any amino acid, preferably V or A <220> <221> VARIOUS_FEATURE <222> (9)..( 9) <223> X is any amino acid, preferably S or T <220> <221> VARIOUS_FEATURE <222> (10)..( 10) <223 > X is any amino acid, preferably S, T or G, <220> <221> VARIOUS_FEATURE <222> (11)..( 11) <223> X is any amino acid, preferably N or H <220 > <221> VARIOUS_FEATURE <222> (14)..( 14) <223> X is any amino acid, preferably G, D or E <400> 3 15 Gly Leu Xaa Ser Gly Ser Xaa Thr Xaa Xaa Xaa Tyr Pro Xaa <210> 316 <211> 14 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <220> <221> VARIOUS_FEATURES <222> (3)..( 3) <223 > X is any amino acid, preferably S, N or I <220> <221> VARIOUS_SPECIAL IK <222> (5)..( 5) <223> X is any amino acid, preferably G or A <220> <221> VARIOUS_FEATURE <222> (6)..( 6) <223> X any an amino acid, preferably N or D <220> <221> VARIOUS_FEATURE <222> (7)..( 7) <223> X is any amino acid, preferably N or S <220> <221> VARIOUS_FEATURE <222> (8)..( 8) <223> X is any amino acid, preferably V or I <220> <221> VARIOUS_FEATURE <222> (10)..( 10) <223> X is any amino acid is preferably N or S <400> 316 Thr Leu xaa Ser xaa xaa xaa xaa Gly xaa Tyr Asp Iie Ser <210> 317 <211>11 <212> PRT <213> Artificial <220> <223> Geim Line Processed Antibody Sequence <220> <221> VARIOUS_FEATURE <222> (6)..( 6) <223> X is any amino acid, preferably G or W <400> 317 Gln Gly Gly Asn Leu Xaa Leu Tyr Gly Ala Asn <210> 318 <211> ll <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <220> <221> VARIOUS FEATURE <222> (4)..( 4) < 223> X any amino acid, preferably S or Tadir <220> <221> VARIOUS_Ö FEATURE <222> (6)..( 6) <223> X is any amino acid, preferably E or Rs <220> <221> VARIOUS_FEATURE <222> (7)..( 7) <223> X is any amino acid, preferably R or N” <220> <221> VARIOUS_FEATURE <222> (9)..( 9) <223> X is any amino acid, preferably G or H7 <220> <221> VARIOUS_FEATURE <222> ( 10)..( 10) <223> X any amino acid, preferably T or A”d1r <400> 318 Arg Gly Asp Xaa Leu Xaa Xaa Tyr Xaa Xaa Asn <210> 319 <211> 14 <212> PRT < 213> Artificial <220> <223> Germline Processed Antibody Sequence <220> <221> VARIOUS_FEATURE <222> (2)..( 2) <223> X is any amino acid, preferably V or L <220 > <221> VARIOUS_FEATURE <222> (3)..( 3) <223> X is any amino acid, preferably K or T <220> <221> VARIOUS_FEATURE <222> (10)..( 10) < 223> X is any amino acid, preferably T or D <400> 319 Gly xaa xaa Ser Gly Ser Vai Thr Ser xaa Asn Phe Pro Thr <210> 320 <211> 10 <212> PRT <213> Homo sapiens < 400> 320 Glu Pro Lys Ser Cys Asp Lys Thr His Thr <210> 321 < 211>5 <212>PRT <213> Homo sapiens <400> 321 <210> 322 <211>8 <212> PRT <213> Homo sapiens <400> 322 Ala Pro Glu Leu Leu Gly Gly Pro <210> 323 < 211> 12 <212> PRT <213> Homo sapiens <400> 323 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr <210> 324 <211> 50 <212> PRT <213> Homo sapiens <400> 324 Votes . ?:0 Arc Cyi Pro Gln Pro good So: Cyi &ip rh: ?:0 Pro ?:0 Cyo l 5 l 0 15 25 30 40 4& <210> 325 <211> 8 <212> PRT <213> Homo sapiens < 400> 325 Ala Pro Glu Leu Leu Gly Gly Pro <210> 326 <211>7 <212> PRT <213> Homo sapiens <400> 326 <210> 327 <21 1> 5 <212> PRT <213> Homo sapiens <400> 327 <210> 328 <211>8 <212> PRT <213> Homo sapiens <400> 328 Ala Pro Glu Phe Leu Gly Gly Pro <210> 329 <211>3 <212> PRT <213> Homo sapiens <400> 329 <210> 330 <211> 10 <212> PRT <213> Homo sapiens <400> 330 Cys Cys Val Glu Cys Pro Pro Pro Cys Pro <210> 331 <211>7 <212> PRT <213> Homo sapiens <400> 331 <210> 332 <211> 142 <212> PRT <213> Homo sapiens <400> 332 1 5 10 15 Gin 610 Rep Pro Arq m ? yt frp Gin Gly Wear Pro Ala ! Main Gly Arc 25 30 End: DM Inn ini aiy Pro is Ldu Mp Lyi aiy ain hu :::9 11. ! lu to (5 Lag up Gly ne 'ryr xit vii &is 11. am vii rh: nu m 11.. Cyi 50 55 60 Sex- Sex- 'mr 'l'hr ! ua Sar Atv His Ilii Pro 'l'hr TI :: neu Ala Va). 613; 65 70 75 80 110 Cyi #8: Pro Ala Sir An; 8.: n. Sor hu Lou kg 140 p.: Ph. 85 90 95 100 105 110 Biy Asp 'nu' uu Cyi 'l'hr un nu 'Hu' Wear TI:: Lou Lou Pro 8.: m 115 120 125 mmriipclurhrrbivhiûiyvi). Gia'rrpvilngvm 130 135 140 <210> 333 <211> 142 <212> PRT <213> Artificial <220> <223> Chimeric Array <400> 333 His Pro aiu Pro Il'u m Ah mu Lou Ola Im: Mu ncu 'mr Vil Pro 1 5 10 15 m by. Asp ?:0 'type 8.: sir 'mr rh:: Ala asi 6.1.0 Üly Cyi ? hr Jun nu- Asp Giu 'i'm <210> 334 <211> 142 <212> PRT <213> Artificial <220> <223> Chimeric Array <400> 334 nn Pro Glu Pro Ilii An; Lyi &ip Pro 'i'hi' 80!' PM 'this' Oiy 01:: Lou 110 Oy. 80: ?to Ala <210> 335 <211> 142 <212> PRT <213> Artificial <220> <223> Chimeric Array <400> 335 80: Prayer ? hr xii aiy 11. Cyi 8.: Pro 11: <210> 336 <211> 142 8.( i 80: Liu <212> PRT <213> Artificial <220> <223> Chimeric Array <400> 336 <210> 337 <211> 142 <212> PRT <213> Artificial <220> <223> Chimeric Array <400> 337 013 cross 110 lt: 80: Arc m 14: up vro rh: 1-00 kg 'up Wear no Oiy Pro ua Lou Wear 25 30 <210> 338 <211> 142 <212> PRT <213> Artificial <220> <223> Chimeric Array <400> 338 3.: ! what ! hr bi. Wear 8.: Sir rh: rh: 110 Cyi 80: !to ki. 81. Bin Giy Cyi Thr 3.: At' 119 Lou 80: &:9 Giy Mp 'Bu' Mu Cyi 'l'hr Ana hu th: Gy ? hr hu Lou ? to 8.: m <210> 339 <211> 142 <212> PRT <213> Artificial <220> <223> Chimeric Array <400> 339 <210> 340 <211> 142 <212> PRT <213> Artificial < 220> <223> Chimeric Array <400> 340 Arc by. Rep Pro 'I'hr m 8.: Sir Pro Gly Sir th: Gin Gin Mp eye i'm- vii m `I'hr Asp Glu Thr Ph. <210> 341 <211> 142 <212> PRT <213> Mus musculus <400> 341 ini Pro Glu Pro Ili. Thr m Lyii np Pro rh: nin 611: Asp Gly Lou 'ryr Arc 611.: 611.1 611 Arc 80:' m 613( Nm 65 70 75 80 11. Cy- sir Pro Ala Al: 8:.: Wear 11. 5 .: Ian Lan Ari; My Arq ! hi 85 90 95 613( mn Mp Oy. 'rhr vu ua Ian Gin Ari; Lou TI:: !yt mi› vii. mi 100 105 110 my up Va). Lou Cyi 'l'hr un Ian 'I'hr Lou Pro Lou ! su !'20 Ask May 11: 120 125 m ni- Asp Glu fb: Ph. Phe Gly V.] 6111 frp what Cys ? to 130 135 130 <210> 342 <211> ll <212> PRT <213> Artificial <220> <223> CD70 Epitope <400> Gln val Thr Leu Ala With Cys Ser Ser <210> 343 <211> 32 <212> PRT <213> Llama glama <400> 343 1 5 10 15 25 30 <210> 344 <211> 1425 <212> DNA <213> Artificial <220> <223> Livelihood Transformed Nucleotide Sequence <400 > 344 tactitgciq iaiqtggiic giaciqtiii otqiitqqca .900909009 ccitccqaui <210> 345 <211> 717 gtocaoecic ÇUIIÇUÜÜCt iutqaacaq citqgaactc qtoqactcti ttwaqtcvi ccciqaagtc <212> DNA <213> Yapay <220> cqqegocttq caqcatoiot tgiciiqm 3:99919919 açgtcucct 90.600.900 ctqtttctqq tOICCICÜVC C'CÜQUOOCC esmer-:99 tac-acemi metne Oaqûaclcüû aciictqgtq ucaoctqcc uttcaciaoq açtgttctqi aqouoim 090%th UUUVOCIUQU CIOtOOGOIO qcciqqtbia ctgiqiacia <223> Germ Hatti Islemi Yapilmis Nükleotit Dizisi <400> 345 ete-ucan tctqotgitt toctitaoiq qqgqtictat cicoqtggit ogoociqcit. aaqctcioat ottqagttcq aqtigttatc <210> 346 <211> 369 Clqtqütglc <212> DNA <213> Yapay <220> icoccgittq cciictqaco aqtiiiciqo cqgiaqtctc Aiqccqcoct tgttcatiiq aciqiagtti <223> Germ Hatti Islemi Yapilmis Nükleotit Dizisi <400> 346 cciqoiiioq oqctiqCth egzotcicqt altili-cet icqitoqteo CCC“. CILÇC MCMO.. iccwiqqic . MMOC ittictqtqo <210>347 <211>341 <212> DNA <213> Artificial <220> <223> Germline Processed Nucleotide Sequence igqgieoqtc oicttqqtac <400> 347 motomcc ! Mein qtctqtooct <210> 348 <211> 372 <212> DNA <213> Artificial <220> tooaqtuct accctqcttq otiocmaq tacucicii iciqccqtci ctctqmtc <223> Germ-Line Processed Nucleotide Sequence OWNcamtqtonict: -um MW? mctcwqt mtatqciq ictocqtçui mccqittc holder: intqaiciiq ::wanna .coqtctece ci <210> 349 <211> 333 <212> DNA <213> Artificial <220> ttwtgcigc cttwtgcigc ctqaiciiq ::wanna .coqtctece ci <210> 349 <211> 333 <212> DNA <213> Artificial <220> ttwtgcigc cttwtgwtc tctqqicmgwtc tctqqicqatctoo tctqqqatoctom: qamatocqattocmt: qamatocqatoctomatc tctqicqattocmt: atamaz& mumu qioccawtc <223> Genn Line Processed Nucleotide Sequence <400> 349 tcctotociq cani-.wow tqitqttm tutusu-.act itqtctcctq qtatcucnq 120 <210> 350 <21 1> 32 <212> <> PRT220213 223> Live Line Processed Antibody Array <400> 350 Am ?hi 'mr ne Sir ::0 Mp m 8.: by: &an 'rhr Mu m :nu Sin 1 5 10 1.5 25 30 <210> 351 <211> 32 <212> PRT <213> Artificial <220> <223> Germline Processed Antibody Array <400> 351

Claims (13)

REQUESTS 1. An antibody or antigen-binding fragment thereof that binds to human CD70, said antibody or antigen-binding fragment being a variable heavy chain CDR3; at least one heavy chain variable domain (VH) containing one variable heavy chain CDR2 and one variable heavy chain CDR1 and at least one light chain variable domain (VL) containing one variable light chain CDR3, one variable light chain CDR2 and one variable light chain CDR1 contains: where variable heavy chain CDR3 SEQ ID. NO:50a (DAGYSNHVPIF DS); variable heavy chain CDR2 SEQ ID. NO:27, (DINNEGGTTYYADSVKG); variable heavy chain CDRl SEQ ID. NO:11” (VYYMN); variable light chain CDR3 SEQ ID. NO: includes or consists of; variable light chain CDR2 SEQ ID. NO: includes or consists of; and the variable light chain CDR1 SEQ ID. NO: contains or consists of. 2. The isolated antibody or antigen-binding fragment of claim 1, SEQ ID. or a heavy chain variable domain (VH) containing or consisting of a 99% identical amino acid sequence and SEQ ID. It contains the amino acid sequence designated NO:241 or a light chain variable domain (VL) composed of them. 3. The isolated antibody or antigen-binding fragment of claim 2, SEQ ID. A heavy chain variable domain (VH) comprising or consisting of the amino acid sequence shown as No:223 and SEQ ID. It contains a light chain variable domain (VL) comprising or consisting of the amino acid sequence shown as No:241. 4. An antibody as claimed in any preceding claim, a llama-human chimeric antibody in which the VH and VL domains are derived from a llama and one or more of the constant domains are derived from a human immunoglobulin, optionally to a human IgG, preferably a human IGG1. It contains the hinge region of ”, the CH2 domain and the CH3 domain. The antibody or antigen-binding fragment of any preceding claim, wherein the antibody or antigen-binding fragment to cells expressing human CD70 on the cell surface, eg, cancer cells expressing CD70, optionally exhibits enhanced ADCC function compared to an equivalent antibody containing a native human Fc domain. exhibits one or more effector functions selected from antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP). An antibody according to any preceding claim, which is a non-fucosylated IgG, preferably a non-fucosylated human IgG17. 7. An isolated polynucleotide encoding the antibody or antigen-binding fragment of any preceding claim. 8. An expression vector comprising the polynucleotide of claim 7 operably linked to regulatory sequences that enable expression of the antigen-binding polypeptide in a host cell or cell-free expression system. 9. A host cell or cell-free expression comprising the expression vector of claim 8 10. A method for producing a recombinant antibody or antigen-binding fragment thereof, comprising expression of the antibody or antigen-binding fragment in the host cell or cell-free expression system of claim 9 and culturing under conditions that allow recovery of the expressed antibody or antigen-binding fragment. 11. A pharmaceutical composition comprising an antibody or antigen-binding fragment of any one of claims 1 to 6 and at least one pharmaceutically acceptable carrier or excipient. The antibody or antigen-binding fragment of any one of claims 1 to 69, which (a) inhibits the growth of CD70-expressing tumor cells in a human patient; or (b) it is used to treat or prevent cancer in a human patient; wherein said cancer is a cancer that optionally exhibits low copy number CD70 expression. 13. The antibody or antigen-binding fragment of any one of claims 1 to 6 for use in the treatment or prevention of cancer in a human patient, wherein the cancer is large B-cell lymphoma, chronic lymphocytic leukemia, Burkitt lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, pancreatic carcinoma, cutaneous T-cell lymphoma, gastric cancer, lung cancer, melanoma, glioblastoma and ovarian cancer.